Enhanced Fano resonances in a silicon nitride photonic crystal nanobeam-assisted micro ring resonator for dual telecom band operation.

Silicon-based Micro Ring Resonators (MRR) are a powerful tool for the realization of label free optical biosensors. The sharp edge of a Fano resonance in a Silicon Nitride (Si3N4) platform can boost photonic sensing applications based on MRRs. In this work, we demonstrate enhanced Fano resonance features for a Si3N4 Micro Ring Resonator assisted by a Photonic Crystal Nanobeam (PhCN-MRR) operating in the TM-like mode at the O-band wavelengths. Our findings show that the fabricated PhCN-MRR results in increased asymmetric resonances for TM-like mode compared with TE-like mode operation in the C-band. As a result, a versatile and flexible design to realize Fano resonance with polarization dependent asymmetry in the C and O telecom bands is presented.

EXPRESS: Mid-Infrared Dispersion Spectroscopy as a Tool for Monitoring Time-Resolved Chemical Reactions on the Examples of Enzyme Kinetics and Mutarotation of Sugars.

Ongoing technological advancements in the field of mid-infrared (MIR) spectroscopy continuously yield novel sensing modalities, offering capabilities beyond traditional techniques like Fourier transform infrared spectroscopy (FT-IR). One such advancement is MIR dispersion spectroscopy, utilizing a tunable quantum cascade laser and Mach-Zehnder interferometer for liquid-phase analysis. Our study assesses the performance of a custom MIR dispersion spectrometer at its current development stage, benchmarks its performance against FT-IR, and validates its potential for time-resolved chemical reaction monitoring. Unlike conventional methods of IR spectroscopy measuring molecular absorptions using intensity attenuation, our method detects refractive index changes (phase shifts) down to a level of 6.1 × 10-7 refractive index units (RIU). This results in 1.5 times better sensitivity with a sevenfold increase in analytical path length, yielding heightened robustness for the analysis of liquids compared to FT-IR. As a case study, we monitor the catalytic activity of invertase with sucrose, observing the formation of resultant monosaccharides and their progression toward thermodynamic equilibrium. Anomalous refractive index spectra of reaction mixtures, with substrate concentrations ranging from 2.5 to 25 g/L, are recorded, and analyzed at various temperatures, yielding Michaelis-Menten kinetics findings comparable to the literature. Additionally, the first-time application of two-dimensional correlation spectroscopy on the recorded dynamic dispersion spectra correctly identifies the mutarotation of reaction products (glucose and fructose). The results demonstrate high precision and sensitivity in investigating complex time-dependent chemical reactions via broadband refractive index changes.

Micro-Ring Resonator Assisted Photothermal Spectroscopy of Water Vapor.

We demonstrated, for the first time, micro-ring resonator assisted photothermal spectroscopy measurement of a gas phase sample. The experiment used a telecoms wavelength probe laser that was coupled to a silicon nitride photonic integrated circuit using a fibre array. We excited the photothermal effect in the water vapor above the micro-ring using a 1395 nm diode laser. We measured the 1f and 2f wavelength modulation response versus excitation laser wavelength and verified the power scaling behaviour of the signal.

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.

Mid-Infrared Photothermal Spectroscopy for the Detection of Caffeine in Beverages.

Caffeine is the most widely consumed stimulant and is the subject of significant ongoing research and discussions due to its impact on human health. The industry's need to comply with country-specific food and beverage regulations underscores the importance of monitoring caffeine levels in commercial products. In this study, we propose an alternative technique for caffeine analysis that relies on mid-infrared laser-based photothermal spectroscopy (PTS). PTS exploits the high-power output of the quantum cascade laser (QCL) sources to enhance the sensitivity of the mid-IR measurement. The laser-induced thermal gradient in the sample scales with the analytes' absorption coefficient and concentration, thus allowing for both qualitative and quantitative assessment. We evaluated the performance of our experimental PTS spectrometer, incorporating a tunable QCL and a Mach-Zehnder interferometer, for detecting caffeine in coffee, black tea, and an energy drink. We calibrated the setup with caffeine standards (0.1-2.5 mg mL-1) and we benchmarked the setup's capabilities against gas chromatography (GC) and Fourier-transform infrared (FTIR) spectroscopy. Quantitative results aligned with GC analysis, and limits of detection matched the research-grade FTIR spectrometer, indicating an excellent performance of our custom-made instrument. This method offers an alternative to established techniques, providing a platform for fast, sensitive, and non-destructive analysis without consumables as well as with high potential for miniaturization.

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.

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.

Simple approach for spectral beam combination of narrowband laser sources for spectroscopic applications.

Spectral beam combination of multiple single mode laser sources employing narrowband spectral filters which are arranged on the perimeter of regular polygons is demonstrated. With this simple geometric design, co-alignment and co-propagation of the individual laser beams can be reasonably achieved. Spectroscopic applicability is displayed by spatial filtering, mode-matching, and subsequent coupling of the combined beams into a 76 m astigmatic mirror multipass cell.

Comparing the direct assessment of steatosis in liver explants with mid- and near-infrared vibrational spectroscopy, prior to organ transplantation.

A fast and accurate assessment of liver steatosis is crucial during liver transplantation surgery as it can negatively impact its success. Recent research has shown that near-infrared (NIR) and attenuated total reflectance-Fourier transform mid-infrared (ATR-FTIR) spectroscopy could be used as real-time quantitative tools to assess steatosis during abdominal surgery. Here, in the frame of a clinical study, we explore the performance of NIR and ATR-FTIR spectroscopy for the direct assessment of steatosis in liver tissues. Results show that both NIR and ATR-FTIR spectroscopy are able to quantify the % of steatosis with cross-validation errors of 1.4 and 1.6%, respectively. Furthermore, the two portable instruments used both provided results within seconds and can be placed inside an operating room evidencing the potential of IR spectroscopy for initial characterization of grafts in liver transplantation surgery. We also evaluated the complementarity of the spectral ranges through correlation spectroscopy.

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.

Enhancing the accuracy of mid-infrared spectroscopy-based liver steatosis quantification using digital image analysis as a reference.

The assessment of liver steatosis is crucial in both hepatology and liver transplantation (LT) surgery. Steatosis can negatively impact the success of LT. Steatosis is a factor for excluding donated organs for LT, but the increasing demand for transplantable organs has led to the use of organs from marginal donors. The current standard for evaluating steatosis is a semi-quantitative grading based on the visual examination of a hematoxylin and eosin (H&E)-stained liver biopsy, but this method is time-consuming, subjective, and lacks reproducibility. Recent research has shown that infrared (IR) spectroscopy could be used as a real-time quantitative tool to assess steatosis during abdominal surgery. However, the development of IR-based methods has been hindered by the lack of appropriate quantitative reference values. In this study, we developed and validated digital image analysis methods for the quantitation of steatosis in H&E-stained liver sections using univariate and multivariate strategies including linear discriminant analysis (LDA), quadratic DA, logistic regression, partial least squares-DA (PLS-DA), and support vector machines. The analysis of 37 tissue samples with varying grades of steatosis demonstrates that digital image analysis provides accurate and reproducible reference values that improve the performance of IR spectroscopic models for steatosis quantification. A PLS model in the 1810-1052 cm-1 region using first derivative ATR-FTIR spectra provided RMSECV = 0.99%. The gained improvement in accuracy critically enhances the applicability of Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) to support an objective graft evaluation at the operation room, which might be especially relevant in cases of marginal liver donors to avoid unnecessary graft explantation.

Fact-based nutrition for infants and lactating mothers-The NUTRISHIELD study.

Background: Human milk (HM) is the ideal source of nutrients for infants. Its composition is highly variable according to the infant's needs. When not enough own mother's milk (OMM) is available, the administration of pasteurized donor human milk (DHM) is considered a suitable alternative for preterm infants. This study protocol describes the NUTRISHIELD clinical study. The main objective of this study is to compare the % weight gain/month in preterm and term infants exclusively receiving either OMM or DHM. Other secondary aims comprise the evaluation of the influence of diet, lifestyle habits, psychological stress, and pasteurization on the milk composition, and how it modulates infant's growth, health, and development. Methods and design: NUTRISHIELD is a prospective mother-infant birth cohort in the Spanish-Mediterranean area including three groups: preterm infants <32 weeks of gestation (i) exclusively receiving (i.e., >80% of total intake) OMM, and (ii) exclusively receiving DHM, and (iii) term infants exclusively receiving OMM, as well as their mothers. Biological samples and nutritional, clinical, and anthropometric characteristics are collected at six time points covering the period from birth and until six months of infant's age. The genotype, metabolome, and microbiota as well as the HM composition are characterized. Portable sensor prototypes for the analysis of HM and urine are benchmarked. Additionally, maternal psychosocial status is measured at the beginning of the study and at month six. Mother-infant postpartum bonding and parental stress are also examined. At six months, infant neurodevelopment scales are applied. Mother's concerns and attitudes to breastfeeding are registered through a specific questionnaire. Discussion: NUTRISHIELD provides an in-depth longitudinal study of the mother-infant-microbiota triad combining multiple biological matrices, newly developed analytical methods, and ad-hoc designed sensor prototypes with a wide range of clinical outcome measures. Data obtained from this study will be used to train a machine-learning algorithm for providing dietary advice to lactating mothers and will be implemented in a user-friendly platform based on a combination of user-provided information and biomarker analysis. A better understanding of the factors affecting milk's composition, together with the health implications for infants plays an important role in developing improved strategies of nutraceutical management in infant care. Clinical trial registration: https://register.clinicaltrials.gov, identifier: NCT05646940.

Laser-Based Mid-Infrared Spectroscopy for Monitoring Temperature-Induced Denaturation of Bovine Serum Albumin and De-/Stabilization Effects of Sugars.

Stability of high-concentration protein formulations is considered a major challenge in current biopharmaceutical development. In this work, we introduce laser-based mid-infrared (IR) spectroscopy as a versatile technique to study the effect of protein concentration and presence of sugars on the thermal denaturation of the model protein bovine serum albumin (BSA). Many analytical techniques struggle to characterize the complex structural transition that occurs during protein denaturation. To this end, a commercially available laser-based mid-IR spectrometer equipped with a customized flow cell was employed to record IR spectra of BSA in the temperature range of 25-85 °C. The temperature perturbation induces a conformational change from a native α-helical to an intermolecular ß-sheet secondary structure in BSA. Systematic investigation of the concentration dependence of the α-ß transition temperature between 30 and 90 mg mL-1 shows a trend of decreasing denaturation temperatures at higher BSA concentrations. In-depth chemometric analysis by a multivariate curve resolution-alternating least squares (MCR-ALS) analysis of the spectra, suggested the formation of not one but two intermediates in the denaturation of BSA. Subsequently, the impact of sugars on denaturation temperatures was investigated, revealing both stabilizing (trehalose, sucrose, and mannose) and destabilizing (sucralose) effects, illustrating the applicability of this method as an investigative tool for stabilizers. These results highlight the potential and versatility of laser-based IR spectroscopy for analysis of protein stability at high concentrations and varying conditions.

Beyond Karl Fischer titration: a monolithic quantum cascade sensor for monitoring residual water concentration in solvents.

Quality control of liquids is an important part of analytical chemistry. The gold standard for measuring residual water in organic solvents and pharmaceutical applications is Karl Fischer titration. It has a high sensitivity, selectivity and accuracy. The downsides are a time-consuming offline analysis, together with the need for toxic reagents producing waste, and it suffers from poor inter-laboratory reproducibility. In this work, we present a high-performance lab-on-a-chip sensor exploiting mid-IR spectroscopy for liquid sensing. It is operating at 6.1 µm wavelength and is suitable for robust and flexible real-time in situ analysis of the residual water concentration in isopropyl alcohol. This is demonstrated in two experiments. A custom-made 60 µL flow cell is employed to measure only minute amounts of analyte in an inline configuration. In a second approach, the whole sensor is immersed into the analyte to demonstrate sensitive and rapid in situ operation on the millisecond time scale. This is confirmed by the ability for time resolved single water-droplet monitoring, while they are mixed into the liquid sample. We obtain a limit of detection between 120 ppm and 150 ppm with a concentration coverage spanning three orders of magnitude from 1.2 × 10-2%vol to 25%vol for the flow cell and 1.5 × 10-2%vol to 19%vol in the in situ configuration, respectively.

Infrared Spectroscopy for Structure Analysis of Protein Inclusion Bodies.

Infrared (IR) spectroscopy is a widely used technique for evaluation of protein secondary structure. In this chapter, we focus on the application of this analytical technique for analysis of inclusion bodies. After a general introduction to protein analysis by IR spectroscopy, different approaches for spectra acquisition, data processing, and secondary structure evaluation are presented.

Mid-IR dispersion spectroscopy - A new avenue for liquid phase analysis.

Mid-IR dispersion spectroscopy is an attractive, novel approach to liquid phase analysis that extends the possibilities of traditional methods based on the detection of absorption via intensity attenuation. This technique detects inherent refractive index changes (phase shifts) induced by IR light interaction with absorbing matter. In contrast to classic absorption spectroscopy, it provides extended dynamic range, baseline-free detection, constant sensitivity, and inherent immunity to power fluctuation. In this paper, we provide a detailed experimental and theoretical characterization and verification of this method with special focus on broadband liquid sample analysis. For this purpose, we develop a compact benchtop dispersion spectroscopy setup based on an EC-QCL coupled to a Mach-Zehnder interferometer. Phase-locked interferometric detection enables to fully harness the advantages of the technique. By instrument operation in the quadrature point combined with balanced detection, the full immunity towards laser power fluctuations and the environmental noise can be achieved. On the example of ethanol (0.5-50% v/v) dissolved in water, it is experimentally demonstrated that changes of the refractive index function are linearly related to concentration also for strongly absorbing, highly concentrated samples beyond the validity of the Beer-Lambert law. Characterization of the sensitivity and noise behavior indicates that the optimum applicable pathlength for liquid analysis can be extended beyond the ones for absorption spectroscopy. Experimental demonstration of the advantages over classical absorption spectroscopy illuminates the potential of dispersion spectroscopy as upcoming robust and sensitive way of recording IR spectra of liquid samples.

Solvent-Free Lipid Separation and Attenuated Total Reflectance Infrared Spectroscopy for Fast and Green Fatty Acid Profiling of Human Milk.

This study presents the first mid-infrared (IR)-based method capable of simultaneously predicting concentrations of individual fatty acids (FAs) and relevant sum parameters in human milk (HM). Representative fat fractions of 50 HM samples were obtained by rapid, two-step centrifugation and subsequently measured with attenuated total reflection IR spectroscopy. Partial least squares models were compiled for the acquired IR spectra with gas chromatography-mass spectrometry (GC-MS) reference data. External validation showed good results particularly for the most important FA sum parameters and the following individual FAs: C12:0 (R2P = 0.96), C16:0 (R2P = 0.88), C18:1cis (R2P = 0.92), and C18:2cis (R2P = 0.92). Based on the obtained results, the effect of different clinical parameters on the HM FA profile was investigated, indicating a change of certain sum parameters over the course of lactation. Finally, assessment of the method's greenness revealed clear superiority compared to GC-MS methods. The reported method thus represents a high-throughput, green alternative to resource-intensive established techniques.

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.

Recombinant Protein L: Production, Purification and Characterization of a Universal Binding Ligand.

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli.