Optics miniaturization strategy for demanding Raman spectroscopy applications.

Raman spectroscopy provides non-destructive, label-free quantitative studies of chemical compositions at the microscale as used on NASA's Perseverance rover on Mars. Such capabilities come at the cost of high requirements for instrumentation. Here we present a centimeter-scale miniaturization of a Raman spectrometer using cheap non-stabilized laser diodes, densely packed optics, and non-cooled small sensors. The performance is comparable with expensive bulky research-grade Raman systems. It has excellent sensitivity, low power consumption, perfect wavenumber, intensity calibration, and 7 cm-1 resolution within the 400-4000 cm-1 range using a built-in reference. High performance and versatility are demonstrated in use cases including quantification of methanol in beverages, in-vivo Raman measurements of human skin, fermentation monitoring, chemical Raman mapping at sub-micrometer resolution, quantitative SERS mapping of the anti-cancer drug methotrexate and in-vitro bacteria identification. We foresee that the miniaturization will allow realization of super-compact Raman spectrometers for integration in smartphones and medical devices, democratizing Raman technology.

Nitroaromatic explosives' detection and quantification using an attention-based transformer on surface-enhanced Raman spectroscopy maps.

Rapidly and accurately detecting and quantifying the concentrations of nitroaromatic explosives is critical for public health and security. Among existing approaches, explosives' detection with Surface-Enhanced Raman Spectroscopy (SERS) has received considerable attention due to its high sensitivity. Typically, a preprocessed single spectrum that is the average of the entire or a selected subset of a SERS map is used to train various machine learning models for detection and quantification. Designing an appropriate averaging and preprocessing procedure for SERS maps across different concentrations is time-consuming and computationally costly, and the averaging of spectra may lead to the loss of crucial spectral information. We propose an attention-based vision transformer neural network for nitroaromatic explosives' detection and quantification that takes raw SERS maps as the input without any preprocessing. We produce two novel SERS datasets, 2,4-dinitrophenols (DNP) and picric acid (PA), and one benchmark SERS dataset, 4-nitrobenzenethiol (4-NBT), which have repeated measurements down to concentrations of 1 nM to illustrate the detection limit. We experimentally show that our approach outperforms or is on par with the existing methods in terms of detection and concentration prediction accuracy. With the produced attention maps, we can further identify the regions with a higher signal-to-noise ratio in the SERS maps. Based on our findings, the molecule of interest detection and concentration prediction using raw SERS maps is a promising alternative to existing approaches.

Methotrexate Detection in Serum at Clinically Relevant Levels with Electrochemically Assisted SERS on a Benchtop, Custom Built Raman Spectrometer.

Therapeutic drug monitoring (TDM) is an essential clinical practice for optimizing drug dosing, thereby preventing adverse effects of drugs with a narrow therapeutic window, slow clearance, or high interperson pharmacokinetic variability. Monitoring methotrexate (MTX) during high-dose MTX (HD-MTX) therapy is necessary to avoid potentially fatal side effects caused by delayed elimination. Despite the efficacy of HD-MTX treatment, its clinical application in resource-limited settings is constrained due to the relatively high cost and time of analysis with conventional analysis methods. In this work, we developed (i) an electrochemically assisted surface-enhanced Raman spectroscopy (SERS) method for detecting MTX in human serum at a clinically relevant concentration range and (ii) a benchtop, Raman detection system with an integrated potentiostat, software, and data analysis unit that enables mapping of small areas of SERS substrates and quantitative SERS-based analysis. In the assay, by promoting electrostatic attraction between gold-coated nanopillar SERS substrates and MTX molecules in aqueous samples, a detection limit of 0.13 µM with a linear range of 0.43-2 µM was achieved in PBS. The implemented sample cleanup through gel filtration proved to be highly effective, resulting in a similar detection limit (0.55 µM) and linear range (1.81-5 µM) for both PBS and serum. The developed and optimized assay could also be used on the in-house built, Raman device. We showed that MTX detection can be carried out in less than 30 min with the Raman device, paving the way toward the TDM of MTX at the point-of-need and in resource-limited environments.

Design of experiments and design space approaches in the pharmaceutical bioprocess optimization.

The optimization of pharmaceutical bioprocesses suffers from several challenges like complexity, upscaling costs, regulatory approval, leading to the risk of delivering substandard drugs to patients. Bioprocess is very complex and requires the evaluation of multiple components that need to be monitored and controlled in order to attain the desired state when the process ends. Statistical design of experiments (DoE) is a powerful tool for optimizing bioprocesses because it plays a critical role in the quality by design strategy as it is useful in exploring the experimental domain and providing statistics of interest that enable scientists to understand the impact of critical process parameters on the critical quality attributes. This review summarizes selected publications in which DoE methodology was used to optimize bioprocess. The main objective of the critical review was to clearly demonstrate potential benefits of using the DoE and design space methodologies in bioprocess optimization.

Development of a prototype device for near real-time surface-enhanced Raman scattering monitoring of biological samples.

With the fast growth of bioanalytical surface-enhanced Raman scattering (SERS), analytical methods have had to adapt to the complex nature of biological samples. In particular, interfering species and protein adsorption onto the SERS substrates have been addressed by sample preparation steps, such as precipitation or extraction, and by smart SERS substrate functionalisation. These additional handling steps however result in irreversible sample alteration, which in turn prevents sample monitoring over time. A new methodology, that enables near real-time, non-invasive and non-destructive SERS monitoring of biological samples, is therefore proposed. It combines solid SERS substrates, benefitting from liquid immersion resistance for extended periods of time, with an original protein filtering device and an on-field detection by means of a handheld Raman analyser. The protein removal device aims at avoiding protein surface fouling on the SERS substrate. It consists of an ultracentrifugation membrane fixed under a cell culture insert for multi-well plates. The inside of the insert is dedicated to containing biological samples. The solid SERS substrate and a simple medium, without any protein, are placed under the insert. By carefully selecting the membrane molecular weight cutoff, selective diffusion of small analytes through the device could be achieved whereas larger proteins were retained inside the insert. Non-invasive SERS spectral acquisition was then carried out through the bottom of the multi-well plate. The diffusion of a SERS probe, 2-mercaptopyridine, and of a neurotransmitter having a less intense SERS signal, serotonin, were first successfully monitored with the device. Then, the latter was applied to distinguish between subclones of cancerous cells through differences in metabolite production. This promising methodology showed a high level of versatility, together with the capability to reduce cellular stress and contamination hazards.

Evaluation of the analytical performances of two Raman handheld spectrophotometers for pharmaceutical solid dosage form quantitation.

This paper addresses the issue of pharmaceutical solid dosage form quantitation using handheld Raman spectrophotometers. The two spectrophotometers used are designed with different technologies: one allows getting a more representative sampling with the Orbital Raster Scanning technology and the other one allows setting acquisition parameters. The goal was to evaluate which technology could provide the best analytical results. Several parameters were optimized to get the lowest prediction error in the end. The main objective of this study was to evaluate if this kind of instrument would be able to identify substandard medicines. For that purpose, two case-study were explored. At first, a full ICH Q2 (R1) compliant validation was performed for moderate Raman scatterer active pharmaceutical ingredient (API) in a specific formulation. It was successfully validated in the ±15% relative total error acceptance limits, with a RMSEP of 0.85% (w/w). Subsequently, it was interesting to evaluate the influence of excipients when the API is a high Raman scatterer. For that purpose, a multi-formulation model was developed and successfully validated with a RMSEP of 2.98% (w/w) in the best case. These two studies showed that thanks to the optimization of acquisition parameters, Raman handheld spectrophotometers methods were validated for two different case-study and could be applied to identify substandard medicines.

Effect of the functionalisation agent on the surface-enhanced Raman scattering (SERS) spectrum: Case study of pyridine derivatives.

Nowadays, the use of functionalised surface-enhanced Raman scattering (SERS) substrates has become common. These surface modifying agents notably act as Raman reporters, as sensors of biological processes (pH, redox probes) or to increase the sensitivity and/or the specificity of SERS detections. However, the effects of the functionalisation agents are deeply examined in very few studies, even though they can affect the aggregation behaviour of the SERS substrate. Moreover, depending on their concentration and on the pH, their spectral signature can be modified and they can even degrade if stored inappropriately. In this context, this paper aims at emphasising the importance of the different aspects previously listed in the selection of a functionalisation agent. Pyridine derivatives were picked out to highlight these parameters, as some of these compounds are commonly used to be grafted onto SERS substrates. Two widespread syntheses of nanoparticles were selected as SERS substrates: citrate-reduced gold and silver nanoparticles. The surface of the nanoparticles was functionalised with several pyridine derivatives at different concentrations and in several solvents. It was observed that the molecules under study had a concentration-dependent effect on nanoparticle aggregation. A stability study was furthermore conducted in order to determine the best preservation conditions of the grafting solutions. In conclusion, this paper shines a light on the relevance of the investigation of the too-often neglected behaviour of the surface modifying agents. Before their application in SERS analyses, parameters such as the label concentration should therefore be included in an experimental design to optimise the sample preparation.

Detection of low dose of piroxicam polymorph in pharmaceutical tablets by surface-enhanced Raman chemical imaging (SER-CI) and multivariate analysis.

This study demonstrates, for the first time, the ability of surface-enhanced Raman chemical imaging (SER-CI) combined with multivariate analysis to detect low levels (0.1% (w/w)) of a polymorphic form in a pharmaceutical mixture. In the studied formulation, piroxicam was used as a model molecule to develop this approach. Piroxicam is a widely available non-steroidal anti-inflammatory drug, exhibiting an interesting case of polymorphism, with two most commonly observed forms (ß and α2). In this work, the SERS spectra of piroxicam polymorphic forms ß and α2 are presented. These forms showed clear spectral differences in terms of band position and intensity. From a crystallographic point of view, the difference of exaltation between both forms was correlated with a preferred orientation of crystallites of form α2 making its SERS detection difficult compared to form ß. A preferred orientation of the (1k0) crystallographic planes of α2 was demonstrated in samples, not promoting an appropriate molecular orientation onto the metallic surface. Additionally, a semi-quantitative approach using SER-CI combined with chemometric tools was developed enabling to detect crystallites of form ß below the detection limit of conventional Raman microscopy. The exaltation of the Raman signal in the presence of silver nanoparticles allowed a higher sensitivity and a reduction of the acquisition time by a factor of 6.

Towards a spray-coating method for the detection of low-dose compounds in pharmaceutical tablets using surface-enhanced Raman chemical imaging (SER-CI).

Surface-enhanced Raman chemical imaging (SER-CI) is a highly sensitive analytical tool recently used in the pharmaceutical field owing to the possibility to obtain high sensitivity along with spatial information. However, the covering method of the pharmaceutical samples such as tablets with metallic nanoparticles is a major issue for SER-CI analyses due to the difficulty to obtain a homogeneous covering of tablet surface with the SERS substrates. In this context, a spray-coating method was proposed in order to fully exploit the potential of SER-CI. A homemade apparatus has been developed from an electrospray ionization (ESI) probe in order to cover the pharmaceutical tablets with the colloidal suspension in a homogeneous way. The silver substrate was pulled through the airbrush by a syringe pump which was then nebulized into small droplets due to the contact of the solution with the gas flow turbulence. A robust optimization of the process was carried out by adjusting experimental parameters such as the liquid flow rate and the spraying time. Besides, the performances of this spraying technique were compared with two others covering methods found in the literature which are drop casting and absorption coating. A homogeneity study, conducted by SER-CI and matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) applied to the different covering techniques was performed. The influence of the metallic nanoparticles deposit on soluble compounds was also investigated in order to highlight the advantages of using this new spray coating approach.

From near-infrared and Raman to surface-enhanced Raman spectroscopy: progress, limitations and perspectives in bioanalysis.

Over recent decades, spreading environmental concern entailed the expansion of green chemistry analytical tools. Vibrational spectroscopy, belonging to this class of analytical tool, is particularly interesting taking into account its numerous advantages such as fast data acquisition and no sample preparation. In this context, near-infrared, Raman and mainly surface-enhanced Raman spectroscopy (SERS) have thus gained interest in many fields including bioanalysis. The two former techniques only ensure the analysis of concentrated compounds in simple matrices, whereas the emergence of SERS improved the performances of vibrational spectroscopy to very sensitive and selective analyses. Complex SERS substrates were also developed enabling biomarker measurements, paving the way for SERS immunoassays. Therefore, in this paper, the strengths and weaknesses of these techniques will be highlighted with a focus on recent progress.