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Background: Surface-enhanced Raman spectroscopy (SERS) has gained increasing importance in molecular detection due to its high specificity and sensitivity. Complex biofluids (e.g., cell lysates and serums) typically contain large numbers of different bio-molecules with various concentrations, making it extremely challenging to be reliably and comprehensively characterized via conventional single SERS spectra due to uncontrollable electromagnetic hot spots and irregular molecular motions. The traditional approach of directly reading out the single SERS spectra or calculating the average of multiple spectra is less likely to take advantage of the full information of complex biofluid systems., Results: Herein, we propose to construct a spectral set with unordered multiple SERS spectra as a novel representation strategy to characterize full molecular information of complex biofluids. This new SERS representation not only contains details from each single spectra but captures the temporal/spatial distribution characteristics. To address the ordering-independent property of traditional chemometric methods (e.g., the Euclidean distance and the Pearson correlation coefficient), we introduce Wasserstein distance (WD) to quantitatively and comprehensively assess the quality of spectral sets on biofluids. WD performs its superiority for the quantitative assessment of the spectral sets. Additionally, WD benefits from its independence of the ordering of spectra in a spectral set, which is undesirable for traditional chemometric methods. With experiments on cell lysates and human serums, we successfully achieve the verification for the reproducibility between parallel samples, the uniformity at different positions in the same sample, the repeatability from multiple tests at one location of the same sample, and the cardinality effect of the spectral set. SERS spectral sets also manage to distinguish different classes of human serums and achieve higher accuracy than the traditional prostate-specific antigen in prostate cancer classification., Significance: The proposed SERS spectral set is a robust representation approach in accessing full information of biological samples compared to relying on a single or averaged spectra in terms of reproducibility, uniformity, repeatability, and cardinality effect. The application of WD further demonstrates the effectiveness and robustness of spectral sets in characterizing complex biofluid samples, which extends and consolidates the role of SERS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Molecular phenotypic variations in metabolites offer the promise of rapid profiling of physiological and pathological states for diagnosis, monitoring, and prognosis. Since present methods are expensive, time-consuming, and still not sensitive enough, there is an urgent need for approaches that can interrogate complex biological fluids at a system-wide level. Here, we introduce hyperspectral surface-enhanced Raman spectroscopy (SERS) to profile microliters of biofluidic metabolite extraction in 15 min with a spectral set, SERSome, that can be used to describe the structures and functions of various molecules produced in the biofluid at a specific time via SERS characteristics. The metabolite differences of various biofluids, including cell culture medium and human serum, are successfully profiled, showing a diagnosis accuracy of 80.8% on the internal test set and 73% on the external validation set for prostate cancer, discovering potential biomarkers, and predicting the tissue-level pathological aggressiveness. SERSomes offer a promising methodology for metabolic phenotyping., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Intracellular metabolic profiling reveals real-time metabolic information useful for the study of underlying mechanisms of cells in particular conditions such as drug resistance. However, mass spectrometry (MS), one of the leading metabolomics technologies, usually requires a large number of cells and complex pretreatments. Surface enhanced Raman scattering (SERS) has an ultrahigh detection sensitivity and specificity, favorable for metabolomics analysis. However, some targeted SERS methods focus on very limited metabolite without global bioprofiling, and some label-free approaches try to fingerprint the metabolic response based on whole SERS spectral classification, but comprehensive interpretation of biological mechanisms was lacking. (95) RESULTS: We proposed a label-free SERS technique for intracellular metabolic profiling in complex cellular lysates within 3 min. We first compared three kinds of cellular lysis methods and sonication lysis shows the highest extraction efficiency of metabolites. To obtain comprehensive metabolic information, we collected a spectral set for each sample and further qualified them by the Pearson correlation coefficient (PCC) to calculate how many spectra should be acquired at least to gain the adequate information from a statistical and global view. In addition, according to our measurements with 10 pure metabolites, we can understand the spectra acquired from complex cellular lysates of different cell lines more precisely. Finally, we further disclosed the variations of 22 SERS bands in enzalutamide-resistant prostate cancer cells and some are associated with the androgen receptor signaling activity and the methionine salvage pathway in the drug resistance process, which shows the same metabolic trends as MS. (149) SIGNIFICANCE: Our technique has the capability to capture the intracellular metabolic fingerprinting with the optimized lysis approach and spectral set collection, showing high potential in rapid, sensitive and global metabolic profiling in complex biosamples and clinical liquid biopsy. This gives a new perspective to the study of SERS in insightful understanding of relevant biological mechanisms. (54)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The detection of deep-seated lesions is of great significance for biomedical applications. However, due to the strong photon absorption and scattering of biological tissues, it is challenging to realize in vivo deep optical detections, particularly for those using the safe laser irradiance below clinical maximum permissible exposure (MPE). In this work, the combination of ultra-bright surface-enhanced Raman scattering (SERS) nanotags and transmission Raman spectroscopy (TRS) is reported to achieve the non-invasive and photosafe detection of "phantom" lesions deeply hidden in biological tissues, under the guidance of theoretical calculations showing the importance of SERS nanotags' brightness and the expansion of laser beam size. Using a home-built TRS system with a laser power density of 0.264 W cm -2 (below the MPE criteria), we successfully demonstrated the detection of SERS nanotags through up to 14-cm-thick ex vivo porcine tissues, as well as in vivo imaging of "phantom" lesions labeled by SERS nanotags in a 1.5-cm-thick unshaved mouse under MPE. This work highlights the potential of transmission Raman-guided identification and non-invasive imaging toward clinically photosafe cancer diagnoses., (© 2022 Wiley-VCH GmbH.)

Self-assembled monolayers (SAMs) of thiols on plasmonic nanoparticles constitute one of the most common methods for fabricating surface-enhanced Raman scattering (SERS) nanoprobes with wide applications. However, this method greatly limits the sufficient utilization of electromagnetic fields derived from plasmon excitation of the nanoparticles, because the thickness of SAMs (<1 nm) is usually much smaller than the attenuation length (>10 nm) of the fields. To overcome this, we propose a three-dimensional (3D) volume-active SERS (VASERS) technique to break the SAM limit, which integrates large amounts of thiol reporters into polydopamine shells on silver nanoparticles via Michael addition and allows sufficient utilization of 3D electromagnetic fields, leading to a dramatic increase in the intensity of the signal of the nanoprobes by about one order of magnitude. We demonstrate the universality of this strategy on various thiol reporters and plasmonic substrates. We also show that orthogonal VASERS nanoprobes with alkyne readout allow for high-precision in vivo tumor targeting and margin delineation.

Surface-enhanced Raman scattering (SERS) nanoprobes have attracted particular interests in the field of bioimaging owing to their high sensitivity and specificity of the fingerprint spectrum. However, the limited signal-to-background ratio (SBR) in SERS imaging and the requirement to perform imaging in a dark environment have largely hindered its biomedical application. To circumvent this, we have developed a type of bio-orthogonal nanoprobes for SERS imaging with an ultrahigh SBR and ambient light anti-interference ability. The core-shell nanoprobes exhibit strongly enhanced Raman signals and depress the background from photoluminescence of metallic nanoparticles by off-resonance excitation and from the Raman scattering and auto-fluorescence of tissues by near-infrared laser excitation. Such nanoprobes have achieved an SBR of over 100 in SERS bioimaging, 5 times higher than the traditional on-resonant nanoprobes, and their bio-orthogonal signal in the Raman-silent region renders the anti-interference capability under ambient light. The development of these SERS probes opens up a new era for the future applications of Raman imaging in clinical medicine.