Label-Free Screening of Drug-Induced Liver Injury Using Stimulated Raman Scattering Microscopy and Spectral Phasor Analysis.

Hepatic toxicity is a leading cause of the termination of clinical trials and the withdrawal of therapeutics following regulatory approval. The detection of drug-induced liver injury (DILI) is therefore of importance to ensure patient safety and the effectiveness of novel small molecules and drugs. DILI encompasses drug-induced steatosis (DIS) and drug-induced phospholipidosis (DIPL) which involve the accumulation of excess intracellular lipids. Here, we develop hyperspectral stimulated Raman scattering (SRS) microscopy as a label-free methodology for discriminating DIS and DIPL in mammalian cell culture. We demonstrate that hyperspectral SRS imaging in tandem with spectral phasor analysis is capable of discriminating DIS and DIPL based on the nature and distribution of intracellular lipids resulting from each process. To demonstrate the practical application of this methodology, we develop a panel of alkyne-tagged propranolol analogues that display varying DILI effects. Using hyperspectral SRS imaging together with spectral phasor analysis, our label-free methodology corroborated the standard fluorescence-based assay for DILI. As a label-free screening method, it offers a convenient and expedient methodology for visualizing hepatotoxicity in cell cultures which could be integrated into the early stages of the drug development process for screening new chemical entities for DILI.

Spectral fingerprinting of cellular lipid droplets using stimulated Raman scattering microscopy and chemometric analysis.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful method for direct visualisation and compositional analysis of cellular lipid droplets. Here we report the application of spectral phasor analysis as a convenient method for the segmentation of lipid droplets using the hyperspectral SRS spectrum in the high wavenumber and fingerprint region of the spectrum. Spectral phasor analysis was shown to discriminate six fatty acids based on vibrational spectroscopic features in solution. The methodology was then applied to studying fatty acid metabolism and storage in a mammalian cancer cell model and during drug-induced steatosis in a hepatocellular carcinoma cell model. The accumulation of fatty acids into cellular lipid droplets was shown to vary as a function of the degree of unsaturation, whilst in a model of drug-induced steatosis, the detection of increased saturated fatty acid esters was observed. Taking advantage of the fingerprint and high wavenumber regions of the SRS spectrum has yielded a greater insight into lipid droplet composition in a cellular context. This approach will find application in the label-free profiling of intracellular lipids in complex disease models.

Multiplex detection of the big five carbapenemase genes using solid-phase recombinase polymerase amplification.

Five carbapenemase enzymes, coined the 'big five', have been identified as the biggest threat to worldwide antibiotic resistance based on their broad substrate affinity and global prevalence. Here we show the development of a molecular detection method for the gene sequences from the five carbapenemases utilising the isothermal amplification method of recombinase polymerase amplification (RPA). We demonstrate the successful detection of each of the big five carbapenemase genes with femtomolar detection limits using a spatially separated multiplex amplification strategy. The approach uses tailed oligonucleotides for hybridisation, reducing the complexity and cost of the assay compared to classical RPA detection strategies. The reporter probe, horseradish peroxidase, generates the measureable output on a benchtop microplate reader, but more notably, our study leverages the power of a portable Raman spectrometer, enabling up to a 19-fold enhancement in the limit of detection. Significantly, the development approach employed a solid-phase RPA format, wherein the forward primers targeting each of the five carbapenemase genes are immobilised to a streptavidin-coated microplate. The adoption of this solid-phase methodology is pivotal for achieving a successful developmental pathway when employing this streamlined approach. The assay takes 2 hours until result, including a 40 minutes RPA amplification step at 37 °C. This is the first example of using solid-phase RPA for the detection of the big five and represents a milestone towards the developments of an automated point-of-care diagnostic for the big five using RPA.

Live chicken egg embryos as an alternative in vivo tumour model for deep surface enhanced Raman spectroscopy.

Live chicken egg embryos offer new opportunities for evaluation and continuous monitoring of tumour growth for in vivo studies compared to traditional rodent models. Here, we report the first use of surface enhanced Raman scattering (SERS) mapping and surface enhanced spatially offset Raman scattering (SESORS) for the detection and localisation of targeted gold nanoparticles in live chicken egg embryos bearing a glioblastoma tumour.

Label-Free Cytometric Evaluation of Mitosis via Stimulated Raman Scattering Microscopy and Spectral Phasor Analysis.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a robust imaging tool for the analysis of biological systems. Here, we present a unique perspective, a label-free spatiotemporal map of mitosis, by integrating hyperspectral SRS microscopy with advanced chemometrics to assess the intrinsic biomolecular properties of an essential process of mammalian life. The application of spectral phasor analysis to multiwavelength SRS images in the high-wavenumber (HWN) region of the Raman spectrum enabled the segmentation of subcellular organelles based on innate SRS spectra. Traditional imaging of DNA is primarily reliant on using fluorescent probes or stains which can affect the biophysical properties of the cell. Here, we demonstrate the label-free visualization of nuclear dynamics during mitosis coupled with an evaluation of its spectral profile in a rapid and reproducible manner. These results provide a snapshot of the cell division cycle and chemical variability between intracellular compartments in single-cell models, which is central to understanding the molecular foundations of these fundamental biological processes. The evaluation of HWN images by phasor analysis also facilitated the differentiation between cells in separate phases of the cell cycle based solely on their nuclear SRS spectral signal, which offers an interesting label-free approach in combination with flow cytometry. Therefore, this study demonstrates that SRS microscopy combined with spectral phasor analysis is a valuable method for detailed optical fingerprinting at the subcellular level.

New Model for Quantifying the Nanoparticle Concentration Using SERS Supported by Multimodal Mass Spectrometry.

Surface-enhanced Raman scattering (SERS) is widely explored for the elucidation of underlying mechanisms behind biological processes. However, the capability of absolute quantitation of the number of nanoparticles from the SERS response remains a challenge. Here, we show for the first time the development of a new 2D quantitation model to allow calibration of the SERS response against the absolute concentration of SERS nanotags, as characterized by single particle inductively coupled plasma mass spectrometry (spICP-MS). A novel printing approach was adopted to prepare gelatin-based calibration standards containing the SERS nanotags, which consisted of gold nanoparticles and the Raman reporter 1,2-bis(4-pyridyl)ethylene. spICP-MS was used to characterize the Au mass concentration and particle number concentration of the SERS nanotags. Results from laser ablation inductively coupled plasma time-of-flight mass spectrometry imaging at a spatial resolution of 5 µm demonstrated a homogeneous distribution of the nanotags (between-line relative standard deviation < 14%) and a linear response of 197Au with increasing nanotag concentration (R2 = 0.99634) in the printed gelatin standards. The calibration standards were analyzed by SERS mapping, and different data processing approaches were evaluated. The reported calibration model was based on an "active-area" approach, classifying the pixels mapped as "active" or "inactive" and calibrating the SERS response against the total Au concentration and the particle number concentration, as characterized by spICP-MS. This novel calibration model demonstrates the potential for quantitative SERS imaging, with the capability of correlating the nanoparticle concentration to biological responses to further understand the underlying mechanisms of disease models.

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis.

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Light-up split aptamers: binding thermodynamics and kinetics for sensing.

Due to their programmable structures, many aptamers can be readily split into two halves while still retaining their target binding function. While split aptamers are prevalent in the biosensor field, fundamental studies of their binding are still lacking. In this work, we took advantage of the fluorescence enhancement property of a new aptamer named OTC5 that can bind to tetracycline antibiotics to compare various split aptamers with the full-length aptamer. The split aptamers were designed to have different stem lengths. Longer stem length aptamers showed similar dissociation constants (Kd) to the full-length aptamer, while a shorter stem construct showed an 85-fold increase in Kd. Temperature-dependent fluorescence measurements confirmed the lower thermostability of split aptamers. Isothermal titration calorimetry indicated that split aptamer binding can release more heat but have an even larger entropy loss. Finally, a colorimetric biosensor using gold nanoparticles was designed by pre-assembling two thiolated aptamer halves, which can then link gold nanoparticles to give a red-to-blue color change.

Understanding radiation response and cell cycle variation in brain tumour cells using Raman spectroscopy.

Radiation therapy is currently utilised in the treatment of approximately 50% of cancer patients. A move towards patient tailored radiation therapy would help to improve the treatment outcome for patients as the inter-patient and intra-patient heterogeneity of cancer leads to large differences in treatment responses. In radiation therapy, a typical treatment outcome is cell cycle arrest which leads to cell cycle synchronisation. As treatment is typically given over multiple fractions it is important to understand how variation in the cell cycle can affect treatment response. Raman spectroscopy has previously been assessed as a method for monitoring radiation response in cancer cells and has shown promise in detecting the subtle biochemical changes following radiation exposure. This study evaluated Raman spectroscopy as a potential tool for monitoring cellular response to radiation in synchronised versus unsynchronised UVW human glioma cells in vitro. Specifically, it was hypothesised that the UVW cells would demonstrate a greater radiation resistance if the cell cycle phase of the cells was synchronised to the G1/S boundary prior to radiation exposure. Here we evaluated whether Raman spectroscopy, combined with cell cycle analysis and DNA damage and repair analysis (γ-H2AX assay), could discriminate the subtle cellular changes associated with radiation response. Raman spectroscopy combined with principal component analysis (PCA) was able to show the changes in radiation response over 24 hours following radiation exposure. Spectral changes were assigned to variations in protein, specifically changes in protein signals from amides as well as changes in lipid expression. A different response was observed between cells synchronised in the cell cycle and unsynchronised cells. After 24 hours following irradiation, the unsynchronised cells showed greater spectral changes compared to the synchronised cells demonstrating that the cell cycle plays an important role in the radiation resistance or sensitivity of the UVW cells, and that radiation resistance could be induced by controlling the cell cycle. One of the main aims of cancer treatment is to stop the proliferation of cells by controlling or halting progression through the cell cycle, thereby highlighting the importance of controlling the cell cycle when studying the effects of cancer treatments such as radiation therapy. Raman spectroscopy has been shown to be a useful tool for evaluating the changes in radiation response when the cell cycle phase is controlled and therefore highlighting its potential for assessing radiation response and resistance.

Evaluating nanoparticle localisation in glioblastoma multicellular tumour spheroids by surface enhanced Raman scattering.

Glioblastoma multiforme (GBM) is a particularly aggressive and high-grade brain cancer, with poor prognosis and life expectancy, in urgent need of novel therapies. These severe outcomes are compounded by the difficulty in distinguishing between cancerous and non-cancerous tissues using conventional imaging techniques. Metallic nanoparticles (NPs) are advantageous due to their diverse optical and physical properties, such as their targeting and imaging potential. In this work, the uptake, distribution, and location of silica coated gold nanoparticles (AuNP-SHINs) within multicellular tumour spheroids (MTS) derived from U87-MG glioblastoma cells was investigated by surface enhanced Raman scattering (SERS) optical mapping. MTS are three-dimensional in vitro tumour mimics that represent a tumour in vivo much more closely than that of a two-dimensional cell culture. By using AuNP-SHIN nanotags, it is possible to readily functionalise the inner gold surface with a Raman reporter, and the outer silica surface with an antibody for tumour specific targeting. The nanotags were designed to target the biomarker tenascin-C overexpressed in U87-MG glioblastoma cells. Immunochemistry indicated that tenascin-C was upregulated within the core of the MTS, however limitations such as NP size, quiescence, and hypoxia, restricted the penetration of the nanotags to the core and they remained in the outer proliferating cells of the spheroids. Previous examples of MTS studies using SERS demonstrated the incubation of NPs on a 2D monolayer of cells, with the subsequent formation of the MTS from these pre-incubated cells. Here, we focus on the localisation of the NPs after incubation into pre-formed MTS to establish a better understanding of targeting and NP uptake. Therefore, this work highlights the importance for the investigation and translation of NP uptake into these 3D in vitro models.

Utilizing Raman Spectroscopy as a Tool for Solid- and Solution-Phase Analysis of Metalloorganic Cage Host-Guest Complexes.

The host-guest chemistry of coordination cages continues to promote significant interest, not least because confinement effects can be exploited for a range of applications, such as drug delivery, sensing, and catalysis. Often a fundamental analysis of noncovalent encapsulation is required to provide the necessary insight into the design of better functional systems. In this paper, we demonstrate the use of various techniques to probe the host-guest chemistry of a novel Pd2L4 cage, which we show is preorganized to selectively bind dicyanoarene guests with high affinity through hydrogen-bonding and other weak interactions. In addition, we exemplify the use of Raman spectroscopy as a tool for analyzing coordination cages, exploiting alkyne and nitrile reporter functional groups that are contained within the host and guest, respectively.

Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy.

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal-to-noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B-H stretch at 2480-2650 cm-1 , together with a bis-alkyne and deuterated fatty acid can be achieved within the cell-silent region of the Raman spectrum. When coupled to imaging in the high-wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells.

Label-Free Imaging of Lipid Droplets in Prostate Cells Using Stimulated Raman Scattering Microscopy and Multivariate Analysis.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful imaging modality for the analysis of biological systems. Here, we report the application of k-means cluster analysis (KMCA) of multi-wavelength SRS images in the high-wavenumber region of the Raman spectrum as a robust and reliable method for the segmentation of cellular organelles based on the intrinsic SRS spectrum. KMCA has been applied to the study of the endogenous lipid biochemistry of prostate cancer and prostate healthy cell models, while the corresponding SRS spectrum of the lipid droplet (LD) cluster enabled direct comparison of their composition. The application of KMCA in visualizing the LD content of prostate cell models following the inhibition of de novo lipid synthesis (DNL) using the acetyl-coA carboxylase inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), is demonstrated. This method identified a reliance of prostate cancer cell models upon DNL for metabolic requirements, with a significant reduction in the cellular LD content after treatment with TOFA, which was not observed in normal prostate cell models. SRS imaging combined with KMCA is a robust method for investigating drug-cell interactions in a label-free manner.

Noninvasive Detection of Ischemic Vascular Damage in a Pig Model of Liver Donation After Circulatory Death.

BACKGROUND AND AIMS: Liver graft quality is evaluated by visual inspection prior to transplantation, a process highly dependent on the surgeon's experience. We present an objective, noninvasive, quantitative way of assessing liver quality in real time using Raman spectroscopy, a laser-based tool for analyzing biomolecular composition. APPROACH AND RESULTS: A porcine model of donation after circulatory death (DCD) with normothermic regional perfusion (NRP) allowed assessment of liver quality premortem, during warm ischemia (WI) and post-NRP. Ten percent of circulating blood volume was removed in half of experiments to simulate blood recovery for DCD heart removal. Left median lobe biopsies were obtained before circulatory arrest, after 45 minutes of WI, and after 2 hours of NRP and analyzed using spontaneous Raman spectroscopy, stimulated Raman spectroscopy (SRS), and staining. Measurements were also taken in situ from the porcine liver using a handheld Raman spectrometer at these time points from left median and right lateral lobes. Raman microspectroscopy detected congestion during WI by measurement of the intrinsic Raman signal of hemoglobin in red blood cells (RBCs), eliminating the need for exogenous labels. Critically, this microvascular damage was not observed during WI when 10% of circulating blood was removed before cardiac arrest. Two hours of NRP effectively cleared RBCs from congested livers. Intact RBCs were visualized rapidly at high resolution using SRS. Optical properties of ischemic livers were significantly different from preischemic and post-NRP livers as measured using a handheld Raman spectrometer. CONCLUSIONS: Raman spectroscopy is an effective tool for detecting microvascular damage which could assist the decision to use marginal livers for transplantation. Reducing the volume of circulating blood before circulatory arrest in DCD may help reduce microvascular damage.

Recent advances in antibiotic resistance diagnosis using SERS: focus on the "Big 5" challenges.

Antibiotic resistant bacteria constitute a global health threat. It is essential for healthcare professionals to prescribe the correct dose of an effective antibiotic to mitigate the bacterial infection in a timely manner to improve the therapeutic outcomes to the patient and prevent the dissemination of antibiotic resistance. To achieve this, there is a need to implement a rapid and ultra-sensitive clinical diagnosis to identify resistant bacterial strains and monitor the effect of antibiotics. In this review, we highlight the use of surface enhanced Raman scattering (SERS) as a powerful diagnostic technique for bacterial detection and evaluation. Initially, this is viewed through a lens covering why SERS can surpass other traditional techniques for bacterial diagnosis. This is followed by different SERS substrates design, detection strategies that have been used for various bacterial biomarkers, how SERS can be combined with other diagnostic platforms to improve its performance towards the bacterial detection and the application of SERS for antibiotic resistance diagnosis. Finally, the recent progress in SERS detection methods in the last decade for the "Big 5" antibiotic resistant challenges as demonstrators of public health major threats is reviewed, namely: Methicillin-resistant Staphylococcus aureus (MRSA), Carbapenem-resistant Enterobacteriaceae (CRE)/Extended-spectrum beta-lactamases (ESBLs), Mycobacterium tuberculosis (TB), Vancomycin-resistant Enterococcus (VRE) and Neisseria Gonorrhoea (NG). This review provides a comprehensive view of the current state of the art with regard to using SERS for assessing antibiotic resistance with a future outlook on where the field go head in the coming years.

Towards quantitative point of care detection using SERS lateral flow immunoassays.

The rapid detection of biomolecules in a point of care (POC) setting is very important for diagnostic purposes. A platform which can provide this, whilst still being low cost and simple to use, is paper-based lateral flow immunoassays (LFIA). LFIA combine immunology and chromatography to detect a target by forming an immunocomplex with a label which traps them in a test zone. Qualitative analysis can be performed using the naked eye whilst quantitative analysis takes place by measuring the optical signal provided by the label at the test zone. There are numerous detection methods available; however, many suffer from low sensitivity and lack of multiplexing capabilities or are poor at providing POC quantitative analysis. An attractive method to overcome this is to use nanoparticles coated in Raman reporters as the labelled species and to analyse test zones using surface-enhanced Raman scattering (SERS). Due to the wide variety of metal nanoparticles, Raman reporter and laser excitations that are available, SERS-based LFIA have been adapted to identify and quantify multiple targets at once. Large Raman microscopes combined with long mapping times have limited the platform to the lab; however, by transferring the analysis to portable Raman instruments, rapid and quantitative measurements can be taken at the POC without any loss in sensitivity. Portable or handheld SERS-LFIA platforms can therefore be used anywhere, from modern clinics to remote and resource-poor settings. This review will present an overview of SERS-based LFIA platforms and the major recent advancements in multiplexing and portable and handheld detection with an outlook on the future of the platform.

Detection of Estrogen Receptor Alpha and Assessment of Fulvestrant Activity in MCF-7 Tumor Spheroids Using Microfluidics and SERS.

Breast cancer is one of the leading causes of cancer death in women. Novel in vitro tools that integrate three-dimensional (3D) tumor models with highly sensitive chemical reporters can provide useful information to aid biological characterization of cancer phenotype and understanding of drug activity. The combination of surface-enhanced Raman scattering (SERS) techniques with microfluidic technologies offers new opportunities for highly selective, specific, and multiplexed nanoparticle-based assays. Here, we explored the use of functionalized nanoparticles for the detection of estrogen receptor alpha (ERα) expression in a 3D tumor model, using the ERα-positive human breast cancer cell line MCF-7. This approach was used to compare targeted versus nontargeted nanoparticle interactions with the tumor model to better understand whether targeted nanotags are required to efficiently target ERα. Mixtures of targeted anti-ERα antibody-functionalized nanotags (ERα-AuNPs) and nontargeted (against ERα) anti-human epidermal growth factor receptor 2 (HER2) antibody-functionalized nanotags (HER2-AuNPs), with different Raman reporters with a similar SERS signal intensity, were incubated with MCF-7 spheroids in microfluidic devices and spectroscopically analyzed using SERS. MCF-7 cells express high levels of ERα and no detectable levels of HER2. 2D and 3D SERS measurements confirmed the strong targeting effect of ERα-AuNP nanotags to the MCF-7 spheroids in contrast to HER2-AuNPs (63% signal reduction). Moreover, 3D SERS measurements confirmed the differentiation between the targeted and the nontargeted nanotags. Finally, we demonstrated how nanotag uptake by MCF-7 spheroids was affected by the drug fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). These results illustrate the potential of using SERS and microfluidics as a powerful in vitro platform for the characterization of 3D tumor models and the investigation of SERD activity.

Mitokyne: A Ratiometric Raman Probe for Mitochondrial pH.

Mitochondrial pH (pHmito) is intimately related to mitochondrial function, and aberrant values for pHmito are linked to several disease states. We report the design, synthesis, and application of mitokyne 1-the first small molecule pHmito sensor for stimulated Raman scattering (SRS) microscopy. This ratiometric probe can determine subtle changes in pHmito in response to external stimuli and the inhibition of both the electron transport chain and ATP synthase with small molecule inhibitors. In addition, 1 was also used to monitor mitochondrial dynamics in a time-resolved manner with subcellular spatial resolution during mitophagy providing a powerful tool for dissecting the molecular and cell biology of this critical organelle.

Correction: Surface enhanced Raman scattering for the multiplexed detection of pathogenic microorganisms: towards point-of-use applications.

Correction for 'Surface enhanced Raman scattering for the multiplexed detection of pathogenic microorganisms: towards point-of-use applications' by Matthew E. Berry et al., Analyst, 2021, DOI: 10.1039/D1AN00865J.