Classification for breast cancer diagnosis with Raman spectroscopy

In order to promote the development of the portable, low-cost and in vivo cancer diagnosis instrument, a miniature laser Raman spectrometer was employed to acquire the conventional Raman spectra for breast cancer detection in this paper. But it is difficult to achieve high discrimination accuracy. Then a novel method of adaptive weight k-local hyperplane (AWKH) is proposed to increase the classification accuracy. AWKH is an extension and improvement of K-local hyperplane distance nearest-neighbor (HKNN). It considers the features weights of the training data in the nearest neighbor selection and local hyperplane construction stage, which resolve the basic shortcoming of HKNN works well only for small values of the nearest-neighbor. Experimental results on Raman spectra of breast tissues in vitro show the proposed method can realize high classification accuracy.OCIS codes: (170.5660) Raman spectroscopy, (300.6450) Spectroscopy, Raman, (170.6510) Spectroscopy, tissue diagnostics     

Cancer field effects in normal tissues revealed by Raman spectroscopy

It has been demonstrated that the presence of cancer results in detectable changes to uninvolved tissues, collectively termed cancer field effects (CFE). In this study, we directly assessed the ability of Raman microspectroscopy to detect CFE via in-vitro study of organotypic tissue rafts approximating human skin. Raman spectra were measured from both epidermis and dermis after transfer of the rafts to dishes containing adherent cultures of either normal human fibroblasts or fibrosarcoma (HT1080) cells. Principal components analyses allowed discrimination between the groups with 86% classification accuracy in the epidermis and 94% in the dermis. These results encourage further study to evaluate the Raman capacity for detecting CFE as a possible tool for noninvasive screening for tumor presence.     

Molecular analysis of the destruction of articular joint tissues by Raman spectroscopy

ABSTRACT

Introduction

Osteoarthritis (OA) is a highly heterogenous disease influenced by different molecular, anatomic, and physiologic imbalances. Some of the bottlenecks for enhanced diagnosis and therapeutic assessment are the lack of validated biomarkers and early diagnosis tools. In this narrative review, we analyze the potential of Raman spectroscopy (RS) as a label-free optical tool for the characterization of articular joint tissues and its application as a diagnosis tool for OA.     

UV reflectance spectroscopy probes DNA and protein changes in human breast tissues

OBJECTIVE: The absorption spectrum obtained using diffuse reflectance measurements of malignant, fibroadenoma, and normal human breast tissues were studied. The spectral features in the spectrum were assigned to molecular components in the tissues. BACKGROUND DATA: Over the past decade, the methods of fluorescence, excitation, and Raman spectroscopy have been studied as potential noninvasive diagnostic tools. Useful spectroscopic information may be obtained from absorption spectra of tissues as well. However, direct measurement of absorption spectra of tissues by conventional transmission means is complicated by multiple photon scattering in tissues. Diffuse reflectance spectrum offers an indirect way to obtain absorption spectrum. METHODS: Excised malignant, fibroadenoma, and normal breast tissue samples without any treatment were obtained from pathology. Samples were placed in a quartz cuvette. The diffuse reflectance measurements between 250 nm to 650 nm were performed using an automated dual lamp spectrophotometer. The absorption spectra of breast tissues were obtained from the diffuse reflectance measurement. RESULTS: Twenty-one invasive carcinoma, 20 mixed in situ and invasive carcinoma, 14 fibroadenoma, and 39 normal breast tissue samples were studied. The absorption spectra of breast tissues were obtained from diffuse reflectance spectra. Spectral features were assigned to DNA and proteins in human breast tissue. Amplitude of changes averaged over 275 nm to 285 nm and 255 nm to 265 nm and were found to be different for malignant, fibroadenoma, and normal breast tissues. These changes arise from differences in content of protein and DNA. CONCLUSION: The peaks of absorption spectrum derived from diffuse reflectance measurements in the UV region revealed fingerprints from proteins and DNA components. The absorbance in the wavelength ranges of 275-285 nm and 255-265 nm were found to be different for malignant, fibroadenoma, and normal breast tissues. These differences provide a criterion to distinguish malignant from fibroadenoma and normal breast tissues.     

Fluorescence and Raman spectroscopy

Table 2 provides a summary of selected in vivo fluorescence and Raman studies performed in BE. Although the findings from these studies appear promising, these techniques are still under development, and it is anticipated that technological refinements will further enhance their diagnostic accuracy. Ultimately, however, large-scale prospective clinical trials are required to determine their true diagnostic potential in BE and other sites. Ideally, the instrumentation of choice would be a real-time endoscopic system that combines excellent diagnostic accuracy with wide-area sampling. In this regard, fluorescence imaging is most appealing, although a variety of issues remain to be resolved, including the choice between autofluorescence versus drug-induced fluorescence and the problematic distinction between dysplastic (true positive) and confounding background metaplastic fluorescence (false positive), among others. It is also not clear whether exogenous fluorophores are necessary to achieve clinically useful sensitivity and specificity for lesion detection in BE. Point spectroscopic techniques, either fluorescence or Raman scattering, are inherently limited by the small volume of tissue (biopsy specimen size) they sample, but more detailed information can be extracted from the spectra, which may increase diagnostic accuracy. Moreover, it may be that the optimal system will be a combination of multiple optical spectroscopic or imaging techniques (multimodality approach), as suggested by Georgakoudi et al. For instance, a lesion could be detected by fluorescence imaging and its dysplastic nature characterized (graded) by Raman spectroscopy. In this era of cost containment, however, the critical challenge is to demonstrate whether an increase in diagnostic accuracy merits investment in costly technology, regardless of the technique used.     

Raman spectroscopy: a real-time tool for identifying microcalcifications during stereotactic breast core needle biopsies

Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. We present here a Raman spectroscopic tool for detecting microcalcifications in breast tissue based on their chemical composition. We collected ex vivo Raman spectra from 159 tissue sites in fresh stereotactic breast needle biopsies from 33 patients, including 54 normal sites, 75 lesions with microcalcifications and 30 lesions without microcalcifications. Application of our Raman technique resulted in a positive predictive value of 97% for detecting microcalcifications. This study shows that Raman spectroscopy has the potential to detect microcalcifications during stereotactic breast core biopsies and provide real-time feedback to radiologists, thus reducing non-diagnostic and false negative biopsies.     

Non-invasive Raman spectroscopy for time-resolved in-line lipidomics

Oil-producing yeast cells are a valuable alternative source for palm oil production and, hence, may be one important piece of the puzzle for a more sustainable future. To achieve a high-quality product, the lipid composition inside oil-producing yeast cells is a crucial parameter for effective process control. Typically, the lipid composition is determined by off-line gas chromatography. A faster, less cumbersome approach is proposed here, by using non-invasive in-line Raman spectroscopy. A fed-batch fermentation of C. oleaginosus – a well-known oleaginous yeast – is used as model experiment to highlight the potential of Raman spectroscopy for in-line lipidomics. The temporal progression of biomass formation, lipid production and glucose consumption are determined based on PLS-regression models allowing process-relevant information on time to be accessed. Additionally, Gaussian curve fitting was applied to extract increasing and decreasing trends of saturated and unsaturated fatty acids produced by C. oleaginosus throughout the fermentation process.

Oil-producing yeast cells are a valuable alternative source for palm oil production and, hence, may be one important piece of the puzzle for a more sustainable future.     

Structure of benzene from mass-correlated rotational Raman spectroscopy

We present high resolution rotational Raman spectra and derived geometry parameters for benzene. Rotational Raman spectra with sub-5 MHz resolution were obtained via high-resolution mass-correlated rotational alignment spectroscopy. Isotopologue spectra for C₆H₆, ¹³C–C₅H₆, C₆D₆, and ¹³C–C₅D₆ were distinguished through their correlated mass information. Spectra for ¹³C₆H₆ were obtained with lower resolution. Equilibrium and effective bond lengths were estimated from measured inertial moments, based on explicit assumptions and approximations. We discuss the origin of significant bias in previously published geometry parameters and the possibility to derive H,D isotope-specific bond lengths from purely experimental data.

We present high resolution rotational Raman spectra and derived geometry parameters for benzene isotopologues. Rotational Raman spectra with sub-5 MHz resolution were obtained via high-resolution mass-correlated rotational alignment spectroscopy.     

Combined total internal reflection AF spectral-imaging and Raman spectroscopy for fast assessment of surgical margins during breast cancer surgery

The standard treatment for breast cancer is surgical removal mainly through breast-conserving surgery (BCS). We developed a new technique based on auto-fluorescence (AF) spectral imaging and Raman spectroscopy for fast intraoperative assessment of excision margins in BCS. A new wide-field AF imaging unit based on total internal reflection (TIR) was combined with a Raman spectroscopy microscope equipped with a 785 nm laser. The wavelength of the AF excitation was optimized to 365 nm in order to maximize the discrimination of adipose tissue. This approach allows for the non-adipose regions of tissue, which are at a higher risk of containing a tumor, to be targeted more efficiently by the Raman spectroscopy measurements. The integrated TIR-AF-Raman was tested on small tissue samples as well as fresh wide local excisions, delivering the analysis of the entire cruciate surface of BCS specimens (5.1 × 7.6 cm²) in less than 45 minutes and also providing information regarding the location of the tumor in the specimen. Full automation of the instrument and selection of a faster translation stage would allow for the measurement of BCS specimens within an intraoperative time scale (20 minutes). This study demonstrates that the TIR-AF Raman microscope represents a feasible step towards the development of a technique for intraoperative assessment of large WLE within intraoperative timescales.     

Noninvasive detection of filaggrin gene mutations using Raman spectroscopy

Knowledge of the existence of filaggrin (FLG) gene mutations might be helpful for a subclassification of patients with atopic dermatitis (AD) which can be used to introduce individualized treatments. In this work the filaggrin content in the skin is assessed using Raman spectroscopy and the results are compared to FLG genotyping of Mexican-mestizo patients. Results showed that the 2282del4 and R501X mutations present in the European population but absent in people of Asian or African descent are also present in the Mexican-mestizo population. The results also showed that patients with filaggrin gene mutations presented lower filaggrin concentrations measured using the vector correlation of their skin Raman spectra and a fixed spectrum of pure human recombinant filaggrin, these results indicate that Raman spectroscopy may be used as a noninvasive tool to detect FLG gene mutations.     

Characterization of peptides self-assembly by low frequency Raman spectroscopy

Low Frequency Vibrational (LFV) modes of peptides and proteins are attributed to the lattice vibrations and are dependent on their structural organization and self-assembly. Studies taken in order to assign specific absorption bands in the low frequency range to self-assembly behavior of peptides and proteins have been challenging. Here we used a single stage Low Frequency Raman (LF-Raman) spectrometer to study a series of diastereomeric analogue peptides to investigate the effect of peptides self-assembly on the LF-Raman modes. The structural variation of the diastereomeric analogues resulted in distinct self-assembly groups, as confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) data. Using LF-Raman spectroscopy, we consistently observed discrete peaks for each of the self-assembly groups. The correlation between the spectral features and structural morphologies was further supported by principal component analysis (PCA). The LFV modes provide further information on the degrees of freedom of the entire peptide within the higher order organization, reflecting the different arrangement of its hydrogen bonding and hydrophobic interactions. Thus, our approach provides a simple and robust complementary method to structural characterization of peptides assemblies.

Characterization of structural changes in peptide assemblies by low frequency Raman spectroscopy.     

电流作用后成骨细胞表面增强的拉曼光谱

目的:多种实验已获知弱强度的电流可促进成骨细胞的增殖、分化和钙盐分泌,进而加速骨伤愈合,加快骨组织代谢。利用表面增强拉曼光谱,可在单细胞水平观察电流刺激下成骨细胞质膜蛋白结构的变化。方法:实验于2006-06/08在南开大学泰达应用物理学院完成。出生4d的昆明品系乳鼠6只,体质量约50g,将1×109L-1密度的成骨细胞用2.5g/L胰酶消化下来,置入到5mmol/L葡萄糖溶液(D-Hanks配置)中。做拉曼光谱的时候取出100μL含有成骨细胞的D-Hanks溶液,使一些成骨细胞固定到CaF2载玻片上。然后应用表面增强拉曼光谱技术,采集0.4mA和1.3mA不同电流强度作用下成骨细胞的拉曼光谱,并与电磁场作用前采集的拉曼光谱做对比。结果:①成骨细胞加电流前后,分别得到30个细胞的拉曼光谱,并利用软件origin7.0算出拉曼光谱的平均相对强度。②根据电流作用前10个细胞的拉曼光谱的相对峰值的平均值和电流作用后10个细胞的拉曼光谱的相对峰值的平均值,得出成骨细胞在受电流作用后,拉曼光谱中11个强峰的相对强度有明显的下降,表明在电流的作用后,成骨细胞膜蛋白构象有很大的变化。③表征蛋白质C-N伸缩振动的谱线1132cm-1的相对强度,在受两种电流作用后,分别增加了128.57%、57.14%,表明细胞质膜蛋白从有序状态转变为无序状态。结论:成骨细胞质膜蛋白在经过电流作用后,细胞膜结构发生较大变化,电流对稳定蛋白质二级结构的氢键和盐键有明显的影响。并且预示拉曼光谱有做为一种潜在的研究骨组织疾病诊断工具的可能性。     

Application driven assessment of probe designs for Raman spectroscopy

In vivo Raman spectroscopy has been utilized for the non-invasive, non-destructive assessment of tissue pathophysiology for a variety of applications largely through the use of fiber optic probes to interface with samples of interest. Fiber optic probes can be designed to optimize the collection of Raman-scattered photons from application-dependent depths, and this critical consideration should be addressed when planning a study. Herein we investigate four distinct probe geometries for sensitivity to superficial and deep signals through a Monte Carlo model that incorporates Raman scattering and fluorescence. Experimental validation using biological tissues was performed to accurately recapitulate in vivo scenarios. Testing in biological tissues agreed with modeled results and revealed that microlens designs had slightly enhanced performance at shallow depths (< 1 mm), whereas all of the beampath-modified designs yielded more signal from deep within tissue. Simulation based on fluence maps generated using ray-tracing in the absence of optical scattering had drastically different results as a function of depth for each probe compared to the biological simulation. The contrast in simulation results between the non-scattering and biological tissue phantoms underscores the importance of considering the optical properties of a given application when designing a fiber optic probe. The model presented here can be easily extended for optimization of entirely novel probe designs prior to fabrication, reducing time and cost while improving data quality.     

Coherent anti-Stokes Raman scattering and spontaneous Raman spectroscopy and microscopy of microalgae with nitrogen depletion

Microalgae are extensively researched as potential feedstocks for biofuel production. Energy-rich compounds in microalgae, such as lipids, require efficient characterization techniques to investigate the metabolic pathways and the environmental factors influencing their accumulation. The model green alga Coccomyxa accumulates significant amounts of triacylglycerols (TAGs) under nitrogen depletion (N-depletion). To monitor the growth of TAGs (lipid) in microalgal cells, a study of microalgal cells (Coccomyxa sp. C169) using both spontaneous Raman and coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy were carried out. Spontaneous Raman spectroscopy was conducted to analyze the components in the algal cells, while CARS was carried out to monitor the distribution of lipid droplets in the cells. Raman signals of carotenoid are greater in control microalgae compared to N-depleted cells. Raman signals of lipid droplets appear after N-depletion and its distribution can be clearly observed in the CARS microscopy. Both spontaneous Raman spectroscopy and CARS microscopy were found to be suitable analysis tools for microalgae.OCIS codes: (300.6365) Spectroscopy, (300.6230) Spectroscopy, coherent anti-Stokes Raman scattering, (300.6450) Spectroscopy, Raman     

Raman profile alterations of irradiated human nasopharyngeal cancer cells detected with laser tweezer Raman spectroscopy

Radiotherapy has been widely used for nasopharyngeal carcinoma (NPC) treatment, which causes DNA damage and alterations of macromolecules of cancer cells. However, the Raman profile alterations of irradiated NPC cells remain unclear. In the present study, we used laser tweezers Raman spectroscopy (LTRS) to monitor internal structural changes and chemical modifications in NPC cells after exposure at a clinical dose (2.3 Gy) to X-ray irradiation (IR) at a single-cell level. Two types of NPC cell lines, CNE2 (EBV-negative cell line) and C666-1 (EBV-positive cell line), were used. The Raman spectra of cells before and after radiation treatment were recorded by LTRS. The analysis of spectral differences indicated that the IR caused Raman profile alterations of intracellular proteins, DNA base and lipids. Moreover, by using the multivariate statistical analysis including principal component analysis (PCA) and linear discriminant analysis (LDA) algorithm, an accuracy of 90.0% for classification between CNE2 cells before and after IR could be achieved, which was 10% better than that of C666-1 cells. The results demonstrated that CNE2 cells were more sensitive to IR in comparison to C666-1 cells, providing useful information for creating a treatment strategy in clinical practice. This exploratory study suggested that LTRS combined with multivariate statistical analysis would be a novel and effective tool for evaluating the radiotherapeutic effect on tumor cells, and for detection of the corresponding alterations at the molecular level.

Laser tweezer Raman spectroscopy combined with multivariate statistical analysis was used for evaluating the radiotherapeutic effect on a single tumor cell.     

Detecting polystyrene nanoplastics using filter paper-based surface-enhanced Raman spectroscopy

This work presents a novel filter paper-based method using surface-enhanced Raman spectroscopy (SERS), for detecting polystyrene nanoplastics (PSNPs). The SERS system used a simple mixture of spherical Au nanoparticles (AuNPs) and 20 nm nanoplastics deposited onto a filter paper which offered a detection limit of 10 μg mL⁻¹ with a sample volume of 50 μL, and in a rare case 5.0 μg mL⁻¹ (with four aliquits of 50 μL).

This work presents a novel filter paper-based method using surface-enhanced Raman spectroscopy (SERS), for detecting polystyrene nanoplastics (PSNPs).

The global threat of accumulated plastic pollution at the nanoscale has recently raised concerns from both the scientific^1,2 and regulatory^3,4 communities. The potential impacts include toxicity on human, aquatic and terrestrial ecosystems.^1,5–7 Although their larger analogues, microplastics, have gained more extensive coverage in the literature,¹ the nature of problems presented by nanoplastics (e.g., biological and chemical interactions in the environment) may inherently differ from those of microplastics.² Nanoscale plastic particulates (e.g. ∼20 nm) like polystyrene (PS) can bioaccumulate at the cellular level, interact with chromosomes and DNA, and can prompt a ‘trojan-horse’ toxicity effect with co-contaminants.⁸Despite the recent expanse of nanoplastic toxicology research, the field remains deprived of standardised detection methods which makes building robust risk assessment frameworks challenging.⁹ Currently, the most common analytical methods employed for nanoplastic detection are scanning electron microscopy (SEM) and transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), mass spectrometry (MS) and Raman spectroscopy.¹⁰ Unfortunately, each technique faces resolution limits at the nanoscale and as such more efficient means of observing nanoplastics must be developed.Surface-enhanced Raman spectroscopy (SERS) on a confocal microscope offers a high spatial resolution with minimal sample preparation.¹¹ The SERS technique, in general, uses a nanostructured plasmonic material, typically consisting of gold and/or silver, which can significantly enhance the Raman scattering signals from resonant interactions with analytes. Consequently, SERS compensates for the inherently low Raman scattering cross sections and concentrations of nanoplastics.Reported systems in the literature typically use a SERS active colloidal system for in situ measurements^12,13 or carefully designed nanostructured surfaces.^14,15 While sample preparation for colloidal systems is comparably simpler, the limit of detection (LOD) is considered to be relatively high (∼40 μg mL⁻¹). In contrast, nanostructured surfaces have demonstrated improved effectiveness (LOD = 10 μg mL⁻¹), but the fabrication of such substrates are difficult.This work presents a filter paper-based SERS substrate (Fig. 1(a)) which combines the advantages of colloidal and nanostructured solid systems, boasting a simple production method with good sensitivity. Particular advantages attributed to filter paper-based SERS are the ability to: create greater hot-spot sites, concentrate analyte, and ability to separate non-target molecules (if required).¹⁶ Filter paper-based SERS substrates have recently shown strong capability in detecting trace amount of small molecules¹⁶ while a cellulose-based substrate has been reported as a ESI^† for SERS active NPs.¹⁷ To our knowledge, this study is the first account of incorporating AuNPs on filter paper substrates that demonstrates the SERS capability with polystyrene nanoplastics (PSNPs).Open in a separate windowFig. 1(a) Schematic illustration for the filter paper system developed in this work, (b) representative surface-enhanced Raman spectra for PS(+)20 (1.0 mg mL⁻¹) with AuNP2 and unenhanced for PS(+)20 (200 mg mL⁻¹), and (c) box plots of surface-enhanced Raman intensities at 1004 cm⁻¹ from each AuNP batch.Spherical AuNPs were synthesised via a Reversed Turkevich method.¹⁸ As SERS performance is correlated to nanoparticle monodispersity and shape uniformity, synthetic conditions (e.g., chloroauric acid concentration) were varied to find optimum values (Table S1^†). All AuNP batches showed an intense absorption peak at approximately 518 nm (Fig. S1^†), with AuNP2 having the strongest absorption. Size and shape qualities were assessed using dynamic light scattering (DLS, Table S1^†) and transmission electron microscopy (TEM, Fig. S2^†). Of all batches, AuNP1 and AuNP2 showed the best polydispersity index (PDI) of 0.45 and 0.45, respectively, while shape analysis on AuNPs imaged by TEM (Fig. S2, S3, and Table S1^†) showed more uniform diameter distribution for AuNP2 than AuNP1 (4.2 vs. 6.7 nm). Nevertheless, all the synthesised AuNP batches were highly spherical in shape with little variation across each batch (aspect ratios all close to 1.0).Prior to SERS experiments, control Raman spectra of bulk polystyrene and filter paper were collected to identify their respective vibrational peaks when detecting PSNPs. Notable Raman signals from PS (Fig. S4^†) include the aromatic ring C–C stretching (1004 cm⁻¹), C–H in-plane deformation (1030 cm⁻¹), and aromatic ring C C deformation (620 cm⁻¹).¹⁹ From filter paper (Fig. S2^†), the signals are attributed to cellulose; glycosidic linkage stretching modes (1095 and 1120 cm⁻¹)²⁰ and C–C stretching mode (995 cm⁻¹).²¹ Considering the relative strength of the observed peaks from PS and lack of complete signal overlap (Fig. S4 and S5^†), we selected the Raman peak at 1004 cm⁻¹ as the reference peak for our discussions.Selective enhancement of analyte signals over the filter paper matrix or the capping agent on the AuNP surface is key to the development of successful SERS substrates. The initial set of SERS experiments (Fig. S5^†) using AuNP2 and positively charged PSNPs (PS(+)20) at 1.0 mg mL⁻¹ showed neither a significant change to the characteristic peaks observed in the control samples nor an enhancement of trisodium citrate (capping agent) and filter paper (Fig. S2^†). Additionally, a proportional enhancement was observed for all PS peaks. These observations collectively suggest: 1. AuNPs and PSNPs do not chemically interact with the filter paper matrix; and 2. covalent bonding is not the enhancement mechanism for this system (preferential enhancement of specific vibrational peak is expected otherwise); physisorption and hotspot field creation by AuNPs are likely pathways.To assess SERS performance and calculate the enhancement factor (EF) of each AuNP batch, Raman spectra (Fig. 1(b)) for PS(+)20 (1.0 mg mL⁻¹) mixed with AuNPs (Au concentration fixed at 0.05 mg mL⁻¹) were collected at fixed instrument parameters (e.g., laser power and confocal hole size, details found in the Method and Materials, ESI^†). For the un-enhanced PSNP spectrum, the concentration was increased to 200 mg mL⁻¹ to obtain sufficient signals at 1004 cm⁻¹. The calculated EF (calculation method is shown in ESI^†) for most AuNP batches was within the range of ∼1100–1750, except for AuNP2 with an improved EF of ∼3050 with an interquartile range of 1440 (∼50% variance) for AuNP2 (a list of EF and interquartile ranges for each AuNP is also available in the Table S3^†). The origin of the higher EF of AuNP2 can be attributed to its more uniform diameter distribution of AuNP2 and the strongest plasmon band at 518 nm (Fig. S1^†). Therefore, we, hereafter, only used AuNP2 for further SERS testing.Next, the LOD for PS(+)20 was determined using AuNP2. The Raman spectra of AuNP2 at different PS(+)20 concentrations, varying from 500–1.0 μg mL⁻¹, are presented in Fig. 2. The PS peak at 1004 cm⁻¹, is apparent at concentrations as low as 10 μg mL⁻¹. The broad peak observed at around 1000 cm⁻¹ at 1.0 μg mL⁻¹, indicated an overlap between the vibrational peaks from the PS and the pyranose backbone vibrations of filter paper. To distinguish each contribution, we compared this spectrum bare filter paper with Raman intensities normalised to the peak positioned at 1095 cm⁻¹ (Fig. S6^†). The difference between them was marginal, thus we conclude this system to have a LOD of 10 μg mL⁻¹.Open in a separate windowFig. 2Surface-enhanced Raman spectra for PS(+)20 (concentration varied from 500–1.0 μg mL⁻¹) with AuNP2 on filter paper. Each spectrum is an average of three independent sets of measurements from different spots.Further, we tested the possibility of concentrating analyte by applying multiple aliquots with PS(+)20 (5.0 μg mL⁻¹) and AuNP2. While most sampled spots showed no PS peaks, in a rare case, successful detection of PSNP was observed (Fig. S7 and S8^†). We hypothesise that this enhancement is attributed to the formation of abnormal number of hot spots within the sampled region. Therefore, the one-off result is not representative and the mechanism underpinning such strong enhancement remains unclear (neither is this the scope of this work). However, future studies can reproducibly exploit this phenomenon to achieve even lower detection limit by redesigning (e.g., optimising the AuNP/analyte ratio) and identifying the underlying enhancement mechanism.These SERS experiments were repeated with different size (PS(+)200) and PSNPs bearing different surface charge (negative charge). With an increase to the analyte size, the LOD was significantly reduced to 1.0 mg mL⁻¹ (Fig. S8^†), possibly attributed to the reduced number of PS particles exposed to the SERS active AuNPs (i.e., much of bulk mass of PS particles is not exposed to SERS hotspot). This observation is contrary to the reported trend in other SERS-based nanoplastic detection systems,^13,17 where our detection limit was lower for larger nanoplastics. Furthermore, we observed that the enhancement effect was not apparent for the negatively charged PSNPs, irrespective of particle size (PS(−)20 and PS(−)200) (Fig. S9^†). This quenching effect is likely attributed to the electrostatic repulsion between the AuNPs and PSNPs, preventing PSNPs to be in the SERS hotspot of AuNPs.Last, the effect of NaCl concentration was tested on our SERS system (150 and 600 mM), to approximate ionic strengths of physiologically relevant biological fluid and seawater, respectively. At 100 μg mL⁻¹, the Raman intensities at the 1004 cm⁻¹ were marginally affected due to the presence of NaCl (Fig. S10^†). However, the signal enhancement was completely quenched at 10 μg mL⁻¹ of nanoplastic (Fig. 3) for both 150 and 600 mM, lowering the LOD to 100 μg mL⁻¹.Open in a separate windowFig. 3Surface-enhanced Raman spectra for PS(+)20 (10 μg mL⁻¹) with or without NaCl (150 or 600 mM).While our filter paper-based SERS system presents a robust and efficient detection method, several considerations must be noted in order to improve its performance. First, the SERS effect is significantly quenched when targeting nanoplastics with the surface charge opposite to the AuNPs incorporated in the filter paper matrix. Thus, its future design can include the use of positively charged AuNPs as a second set of testing, for effective detection of negatively charged PSNPs. Similarly, the presence of NaCl quenched the SERS effect indicating future application may require a desalination step (e.g., dialysis) prior to analyte addition onto filter paper and subsequent Raman measurements. Lastly, this study showed the correlation between the improved Raman enhancement and particle size distribution (or the optimum synthetic condition to achieve this). However, to further establish this structure-performance relationship and desirable synthetic conditions, more systematic studies are warranted with a wide range of AuNP size and size distribution.It is also established that nano-particulates in complex environmental and biological fluids are covered with “corona” layer(s) of the pre-existing molecules.¹ The capability of SERS systems (not limited to filter paper-based) to detect both nanoplastics and corona layer (i.e., whether SERS hot-spot can penetrate through the corona layer) is yet to be investigated in depth. Given the importance of identifying the molecular compositions of the corona layer in the risk assessment framework,¹ it is important that future studies will tackle this challenge. Additionally, the future studies can effectively use chemometrics to help eliminating the cellulose background signals and distinguish between different plastic types (and other molecules).In summary, this study demonstrated the first proof of principle for using filter paper as a supporting matrix of SERS active NPs to detect 20 nm sized PSNPs. A reliable LOD was 10 μg mL⁻¹ was achieved, in a rare case, we observed the successful detection of PSNPs at 5 μg mL⁻¹. While strong enhancements (EF = ∼3050 with ∼50% variance) for PS(+)20 was confirmed with AuNP2 (batch of which that showed the most narrow size distribution), the extent of which was significantly reduced for larger nanoplastics (PS(+)200) or completely quenched for negatively charged PSNPs (PS(−)20 and PS(−)200). While uniformity of the SERS matrix is the key to the reproducible and effective enhancement, the present system demonstrates that the heterogenous AuNP/nanoplastic aggregate formation is the essential condition to achieve these. Nevertheless, this simple system offers great potential in detecting nanoplastics and may be a forerunning candidate as a standardised methodology for plastic nanotoxicology research.     

Step-by-step monitoring of CVD-graphene during wet transfer by Raman spectroscopy

Transfer acts as a crucial bridge between the chemical vapor deposition (CVD) synthesis of large-scale graphene and its applications, but the quality evolution of a graphene film during transfer remains unclear. Here we use scanning Raman spectroscopy to monitor as-grown graphene during each step of wet transfer including floating on etchant solution, loaded onto a target substrate, and with additional annealing. Results show that the etchant solution results in strong compressive strain and p-type doping to floating graphene, but both are significantly reduced after the sample is loaded and rinsed especially for the doping. An annealing treatment increases the compressive strain in graphene but hardly its doping level. Moreover, when a poly(methyl methacrylate) (PMMA) layer is used to assist the transfer, it does not only increase the p-type doping of floating graphene but also lowers the crystalline quality of annealed graphene. Therefore, to obtain graphene with better quality, besides the attempts of improving CVD synthesis for its larger domain sizes, universal and easy-to-use polymer-free transfer techniques must be developed as well.

The quality evolution of as-grown graphene during wet transfer from Cu to SiO₂/Si substrate is investigated by Raman spectroscopy and the relavant factors during this process are identified.     

Optical diagnosis of oral cavity lesions by label-free Raman spectroscopy

Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers and frequently preceded by non-malignant lesions. Using Shifted-Excitation Raman Difference Spectroscopy (SERDS), principal component and linear discriminant analysis in native tissue specimens, 9500 raw Raman spectra of OSCC, 4300 of non-malignant lesions and 4200 of physiological mucosa were evaluated. Non-malignant lesions were distinguished from physiological mucosa with a classification accuracy of 95.3% (95.4% sensitivity, 95.2% specificity, area under the curve (AUC) 0.99). Discriminating OSCC from non-malignant lesions showed an accuracy of 88.4% (93.7% sensitivity, 76.7% specificity, AUC 0.93). OSCC was identified against physiological mucosa with an accuracy of 89.8% (93.7% sensitivity, 81.0% specificity, AUC 0.90). These findings underline the potential of SERDS for the diagnosis of oral cavity lesions.