Complete characterization of RNA biomarker fingerprints using a multi-modal ATR-FTIR and SERS approach for label-free early breast cancer diagnosis.

Breast cancer is a prevalent form of cancer worldwide, and the current standard screening method, mammography, often requires invasive biopsy procedures for further assessment. Recent research has explored microRNAs (miRNAs) in circulating blood as potential biomarkers for early breast cancer diagnosis. In this study, we employed a multi-modal spectroscopy approach, combining attenuated total reflection Fourier transform infrared (ATR-FTIR) and surface-enhanced Raman scattering (SERS) to comprehensively characterize the full-spectrum fingerprints of RNA biomarkers in the blood serum of breast cancer patients. The sensitivity of conventional FTIR and Raman spectroscopy was enhanced by ATR-FTIR and SERS through the utilization of a diamond ATR crystal and silver-coated silicon nanopillars, respectively. Moreover, a wider measurement wavelength range was achieved with the multi-modal approach than with a single spectroscopic method alone. We have shown the results on 91 clinical samples, which comprised 44 malignant and 47 benign cases. Principal component analysis (PCA) was performed on the ATR-FTIR, SERS, and multi-modal data. From the peak analysis, we gained insights into biomolecular absorption and scattering-related features, which aid in the differentiation of malignant and benign samples. Applying 32 machine learning algorithms to the PCA results, we identified key molecular fingerprints and demonstrated that the multi-modal approach outperforms individual techniques, achieving higher average validation accuracy (95.1%), blind test accuracy (91.6%), specificity (94.7%), sensitivity (95.5%), and F-score (94.8%). The support vector machine (SVM) model showed the best area under the curve (AUC) characterization value of 0.9979, indicating excellent performance. These findings highlight the potential of the multi-modal spectroscopy approach as an accurate, reliable, and rapid method for distinguishing between malignant and benign breast tumors in women. Such a label-free approach holds promise for improving early breast cancer diagnosis and patient outcomes.

Diffuse reflectance spectroscopy and imaging for non-invasive objective assessment of genitourinary syndrome of menopause: a pilot study.

The genitourinary symptom of menopause (GSM) affects up to 65% of women, resulting in symptoms such as vulvovaginal dryness, discomfort, and dysuria, which significantly impacts quality of life. The current assessment methods rely on subjective questionnaires that can be influenced by individual differences, as well as invasive measurements that are time-consuming and not easily accessible. In this study, we explore the potential of a non-invasive and objective assessment tool called diffuse reflectance spectroscopy and imaging (DRSI) to evaluate tissue chromophores, including water, lipid, oxyhemoglobin, and deoxyhemoglobin. These measurements provide information about moisture content, lipid levels, oxygen saturation, and blood fraction, which can serve as surrogate markers for genital estrogen levels. Our findings reveal distinct differences in these chromophores among pre, peri, and postmenopausal subjects. By using lipid and blood fraction tissue chromophores in a K-Nearest Neighbour classifier model, we achieved a prediction accuracy of 65% compared to vaginal maturation index (VMI) that is clinically used to assess estrogen-related hormonal changes. When age was included as the third feature, the accuracy increased to 78%. We believe that by refining the study protocol and configuring the fiber probe to examine tissue chromophores both in the superficial vulva skin for epidermal water content and the deeper layers, DRSI has the potential to provide objective diagnosis and aid in monitoring the treatment outcome of GSM.

A pilot study on non-invasive in situ detection of phytochemicals and plant endogenous status using fiber optic infrared spectroscopy.

Traditional methods for assessing plant health often lack the necessary attributes for continuous and non-destructive monitoring. In this pilot study, we present a novel technique utilizing a customized fiber optic probe based on attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) with a contact force control unit for non-invasive and continuous plant health monitoring. We also developed a normalized difference mid-infrared reflectance index through statistical analysis of spectral features, enabling differentiation of drought and age conditions in plants. Our research aims to characterize phytochemicals and plant endogenous status optically, addressing the need for improved analytical measurement methods for in situ plant health assessment. The probe configuration was optimized with a triple-loop tip and a 3 N contact force, allowing sensitive measurements while minimizing leaf damage. By combining polycrystalline and chalcogenide fiber probes, a comprehensive wavenumber range analysis (4000-900 cm-1) was achieved. Results revealed significant variations in phytochemical composition among plant species, for example, red spinach with the highest polyphenolic content and green kale with the highest lignin content. Petioles displayed higher lignin and cellulose absorbance values compared to veins. The technique effectively monitored drought stress on potted green bok choy plants in situ, facilitating the quantification of changes in water content, antioxidant activity, lignin, and cellulose levels. This research represents the first demonstration of the potential of fiber optic ATR-FTIR probes for non-invasive and rapid plant health measurements, providing insights into plant health and advancements in quantitative monitoring for indoor farming practices, bioanalytical chemistry, and environmental sciences.

Fiber optic probe-based ATR-FTIR spectroscopy for rapid breast cancer detection: A pilot study.

Breast cancer diagnosis is crucial for timely treatment and improved outcomes. This paper proposes a novel approach for rapid breast cancer diagnosis using optical fiber probe-based attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy from 750 to 4000 cm-1 . The technique enables direct analysis of tissue samples, eliminating the need for microtome sectioning and staining, thus saving time and resources. By capturing molecular fingerprint information, various machine-learning models were used to analyze the spectroscopic data to classify cancerous and non-cancerous tissues accurately. Comparing deparaffinized and paraffinized samples reveals the impact of sample preparation and experimental methods. The study demonstrates a strong correlation between the cancerous nature of a sample and its ATR-FTIR spectrum, suggesting its potential for breast cancer diagnosis (sensitivity of 74.2% and specificity of 78.3%). The proposed approach holds promise for integration into clinical operations, providing a rapid method for preliminary breast cancer diagnosis.

Molecular Fingerprint Detection Using Raman and Infrared Spectroscopy Technologies for Cancer Detection: A Progress Review.

Molecular vibrations play a crucial role in physical chemistry and biochemistry, and Raman and infrared spectroscopy are the two most used techniques for vibrational spectroscopy. These techniques provide unique fingerprints of the molecules in a sample, which can be used to identify the chemical bonds, functional groups, and structures of the molecules. In this review article, recent research and development activities for molecular fingerprint detection using Raman and infrared spectroscopy are discussed, with a focus on identifying specific biomolecules and studying the chemical composition of biological samples for cancer diagnosis applications. The working principle and instrumentation of each technique are also discussed for a better understanding of the analytical versatility of vibrational spectroscopy. Raman spectroscopy is an invaluable tool for studying molecules and their interactions, and its use is likely to continue to grow in the future. Research has demonstrated that Raman spectroscopy is capable of accurately diagnosing various types of cancer, making it a valuable alternative to traditional diagnostic methods such as endoscopy. Infrared spectroscopy can provide complementary information to Raman spectroscopy and detect a wide range of biomolecules at low concentrations, even in complex biological samples. The article concludes with a comparison of the techniques and insights into future directions.

Label-free detection of MiRNA biomarkers using broadband multi-resonant infrared metasurfaces for early breast cancer diagnosis.

Breast cancer is the most prevalent cancer globally. Early detection is crucial and can be achieved by detecting cancer biomarkers in blood, such as circulating miRNAs (microRNAs). In this study, we present a label-free detection method based on broadband multi-resonant infrared metasurface for surface-enhanced infrared absorption (SEIRA) spectroscopy to detect miRNAs. The SEIRA resonances were optimized to match the miRNA biomarker fingerprint regions in the range of 800 to 2000 cm-1 and 2800 to 3500 cm-1, resulting in a simulated resonance enhancement of up to 103 times. Nine patient samples (six cancerous and three non-cancerous) were measured using SEIRA multi-well sensor chips. A novel analysis method, SEIRA-AR, was also developed to benchmark the results against industry standards, such as quantitative reverse transcription polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Our results showed an excellent linear correlation with a Pearson's r value of up to 0.99 and an R Squared value of up to 0.98. This study represents the first use of a SEIRA sensor for biomarker detection on clinical breast cancer samples and introduces an analysis method that produces results comparable to industry standards. Our findings pave the way for routine cancer diagnosis in the future. Additionally, the method discussed can be generalized to other biosensing activities involving two-step binding processes with complementary molecule-capturing agents.

Machine Learning Assisted Real-Time Label-Free SERS Diagnoses of Malignant Pleural Effusion due to Lung Cancer.

More than half of all pleural effusions are due to malignancy of which lung cancer is the main cause. Pleural effusions can complicate the course of pneumonia, pulmonary tuberculosis, or underlying systemic disease. We explore the application of label-free surface-enhanced Raman spectroscopy (SERS) as a point of care (POC) diagnostic tool to identify if pleural effusions are due to lung cancer or to other causes (controls). Lung cancer samples showed specific SERS spectral signatures such as the position and intensity of the Raman band in different wave number region using a novel silver coated silicon nanopillar (SCSNP) as a SERS substrate. We report a classification accuracy of 85% along with a sensitivity and specificity of 87% and 83%, respectively, for the detection of lung cancer over control pleural fluid samples with a receiver operating characteristics (ROC) area under curve value of 0.93 using a PLS-DA binary classifier to distinguish between lung cancer over control subjects. We have also evaluated discriminative wavenumber bands responsible for the distinction between the two classes with the help of a variable importance in projection (VIP) score. We found that our label-free SERS platform was able to distinguish lung cancer from pleural effusions due to other causes (controls) with higher diagnostic accuracy.

Photoacoustic Tomography Appearance of Fat Necrosis: A First-in-Human Demonstration of Biochemical Signatures along with Histological Correlation.

A 50-year-old woman with no past medical history presented with a left anterior chest wall mass that was clinically soft, mobile, and non-tender. A targeted ultrasound (US) showed findings suggestive of a lipoma. However, focal "mass-like" nodules seen within the inferior portion suggested malignant transformation of a lipomatous lesion called for cross sectional imaging, such as MRI or invasive biopsy or excision for histological confirmation. A T1-weighted image demonstrated a large lipoma that has a central fat-containing region surrounded by an irregular hypointense rim in the inferior portion, confirming the benignity of the lipoma. An ultrasound-guided photoacoustic imaging (PA) of the excised specimen to derive the biochemical distribution demonstrated the "mass-like" hypoechoic regions on US as fat-containing, suggestive of benignity of lesion, rather than fat-replacing suggestive of malignancy. The case showed the potential of PA as an adjunct to US in improving the diagnostic confidence in lesion characterization.

Rapid and sensitive detection of ovarian cancer biomarker using a portable single peak Raman detection method.

Raman spectroscopy (RS) is a widely used non-destructive technique for biosensing applications because of its ability to detect unique 'fingerprint' spectra of biomolecules from the vibrational bands. To detect these weak fingerprint spectra, a complex detection system consisting of expensive detectors and optical components are needed. As a result, surface enhanced Raman spectroscopy (SERS) method were used to increase the Raman signal multifold beyond 1012 times. However, complexity of the entire Raman detection system can be greatly reduced if a short wavelength region/unique single spectral band can distinctly identify the investigating analyte, thereby reducing the need of multiple optical components to capture the entire frequency range of Raman spectra. Here we propose the development of a rapid, single peak Raman technique for the detection of epithelial ovarian cancers (EOC)s through haptoglobin (Hp), a prognostic biomarker. Hp concentration in ovarian cyst fluid (OCF) can be detected and quantified using Raman spectroscopy-based in vitro diagnostic assay. The uniqueness of the Raman assay is that, only in the presence of the analyte Hp, the assay reagent undergoes a biochemical reaction that results in product formation. The unique Raman signature of the assay output falls within the wavenumber region 1500-1700 cm-1 and can be detected using our single peak Raman system. The diagnostic performance of our Raman system had 100.0% sensitivity, 85.0% specificity, 100.0% negative predictive value and 84.2% positive predictive value when compared to gold standard paraffin histology in a proof-of-concept study on 36 clinical OCF samples. When compared to blood-based serum cancer antigen 125 (CA125) levels, the Raman system-based assay had higher diagnostic accuracy when compared to CA125, especially in early-stage EOCs.

Biochemical "decoding" of breast ultrasound images with optoacoustic tomography fusion: First-in-human display of lipid and collagen signals on breast ultrasound.

To date, studies which utilized ultrasound (US) and optoacoustic tomography (OT) fusion (US-OT) in biochemical differentiation of malignant and benign breast conditions have relied on limited biochemical data such as oxyhaemoglobin (OH) and deoxyhaemoglobin (DH) only. There has been no data of the largest biochemical components of breast fibroglandular tissue: lipid and collagen. Here, the authors believe the ability to image collagen and lipids within the breast tissue could serve as an important milestone in breast US-OT imaging with many potential downstream clinical applications. Hence, we would like to present the first-in-human US-OT demonstration of lipid and collagen differentiation in an excised breast tissue from a 38-year-old female.

The lateral entorhinal cortex is a hub for local and global dysfunction in early Alzheimer's disease states.

Functional network activity alterations are one of the earliest hallmarks of Alzheimer's disease (AD), detected prior to amyloidosis and tauopathy. Better understanding the neuronal underpinnings of such network alterations could offer mechanistic insight into AD progression. Here, we examined a mouse model (3xTgAD mice) recapitulating this early AD stage. We found resting functional connectivity loss within ventral networks, including the entorhinal cortex, aligning with the spatial distribution of tauopathy reported in humans. Unexpectedly, in contrast to decreased connectivity at rest, 3xTgAD mice show enhanced fMRI signal within several projection areas following optogenetic activation of the entorhinal cortex. We corroborate this finding by demonstrating neuronal facilitation within ventral networks and synaptic hyperexcitability in projection targets. 3xTgAD mice, thus, reveal a dichotomic hypo-connected:resting versus hyper-responsive:active phenotype. This strong homotopy between the areas affected supports the translatability of this pathophysiological model to tau-related, early-AD deficits in humans.

Acceptor engineering of small-molecule fluorophores for NIR-II fluorescence and photoacoustic imaging.

Fluorescence imaging in the second near-infrared window (NIR-II) has been an emerging technique in diverse in vivo applications with high sensitivity/resolution and deep tissue penetration. To date, the design principle of the reported NIR-II organic fluorophores has heavily relied on benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole) (BBTD) as a strong electron acceptor. Here, we report the rational design and synthesis of a NIR-II fluorescent molecule with the rarely used [1,2,5]thiadiazolo[3,4-f]benzotriazole (TBZ) core to replace BBTD as the electron acceptor. Thanks to the weaker electron deficiency of the TBZ core than BBTD, the newly yielded NIR-II molecule (BTB) based nanoparticles have a higher mass extinction coefficient and quantum yield in water. In contrast, the nanoparticle suspension of its counterpart with BBTD as the core is nearly nonemissive. The NIR-II BTB nanoparticles allow video-rate fluorescence imaging for vasculature imaging in ears, hindlimbs, and the brain of the mouse. Additionally, its large absorptivity in the NIR-I region also promotes bioimaging using photoacoustic microscopy (PAM) and tomography (PAT). Upon surface conjugation with the Arg-Gly-Asp (RGD) peptide, the functionalized nanoparticles ensured targeted detection of integrin-overexpressed tumors through both imaging modalities in two- and three-dimensional views. Thus, our approach to engineering acceptors of organic fluorophores offers a promising molecular design strategy to afford new NIR-II fluorophores for versatile biomedical imaging applications.

Magnetic Particle Imaging: An Emerging Modality with Prospects in Diagnosis, Targeting and Therapy of Cancer.

BACKGROUND: Magnetic Particle Imaging (MPI) is an emerging imaging modality for quantitative direct imaging of superparamagnetic iron oxide nanoparticles (SPION or SPIO). With different physics from MRI, MPI benefits from ideal image contrast with zero background tissue signal. This enables clear visualization of cancer with image characteristics similar to PET or SPECT, but using radiation-free magnetic nanoparticles instead, with infinite-duration reporter persistence in vivo. MPI for cancer imaging: demonstrated months of quantitative imaging of the cancer-related immune response with in situ SPION-labelling of immune cells (e.g., neutrophils, CAR T-cells). Because MPI suffers absolutely no susceptibility artifacts in the lung, immuno-MPI could soon provide completely noninvasive early-stage diagnosis and treatment monitoring of lung cancers. MPI for magnetic steering: MPI gradients are ~150 × stronger than MRI, enabling remote magnetic steering of magneto-aerosol, nanoparticles, and catheter tips, enhancing therapeutic delivery by magnetic means. MPI for precision therapy: gradients enable focusing of magnetic hyperthermia and magnetic-actuated drug release with up to 2 mm precision. The extent of drug release from the magnetic nanocarrier can be quantitatively monitored by MPI of SPION's MPS spectral changes within the nanocarrier. CONCLUSION: MPI is a promising new magnetic modality spanning cancer imaging to guided-therapy.

Circulating microRNA breast cancer biomarker detection in patient sera with surface plasmon resonance imaging biosensor.

In this article, we report for the first time, the detection of circulating miRNA as a breast cancer biomarker in patient sera using surface plasmon resonance imaging biosensor. The advantage of this approach lies in the rapid, label-free and sensitive detection. The sensor excites plasmonic resonance on the gold sensor surface and specific DNA-miRNA molecular bindings elucidate responses in the plasmonic resonance image. Experiments of detecting synthetic miRNA molecules (miR-1249) were performed and the sensor resolution was found to be 63.5 nM. The sensor was further applied to screen 17 patient serum samples from National Cancer Centre Singapore and Tan Tock Seng Hospital. Sensor intensity response was found to differ by 20% between malignant and benign cases and thus forms, a potential and an important metric in distinguishing benignity and malignancy.

Biophotonic technologies for assessment of breast tumor surgical margins-A review.

Breast conserving surgery (BCS) offering similar surgical outcomes as mastectomy while retaining breast cosmesis is becoming increasingly popular for the management of early stage breast cancers. However, its association with reoperation rates of 20% to 40% following incomplete tumor removal warrants the need for a fast and accurate intraoperative surgical margin assessment tool that offers cellular, structural and molecular information of the whole specimen surface to a clinically relevant depth. Biophotonic technologies are evolving to qualify as such an intraoperative tool for clinical assessment of breast cancer surgical margins at the microscopic and macroscopic scale. Herein, we review the current research in the application of biophotonic technologies such as photoacoustic imaging, Raman spectroscopy, multimodal multiphoton imaging, diffuse optical imaging and fluorescence imaging using medically approved dyes for breast cancer detection and/or tumor subtype differentiation toward intraoperative assessment of surgical margins in BCS specimens, and possible challenges in their route to clinical translation.

Optoacoustic characterization of breast conserving surgery specimens - A pilot study.

In this pilot study, we tested an ultrasound-guided optoacoustic tomography (US-OT) two-dimensional (2D) array scanner to understand the optoacoustic patterns of excised breastconserving surgery (BCS) specimens. We imaged 14 BCS specimens containing malignant tumors at eight wavelengths spanning 700-1100 nm. Spectral unmixing across multiple wavelengths allowed for visualizing major intrinsic chromophores in the breast tissue including hemoglobin and lipid up to a depth of 7 mm. We identified less/no lipid signals within the tumor and intense deoxy-hemoglobin (Hb) signals on the rim of the tumor as unique characteristics of malignant tumors in comparison to no tumor region. We also observed continuous broad lipid signals as features of negative margins and compromised lipid signals interrupted by vasculature as features of positive margins. These differentiating patterns can form the basis of US-OT to be explored as an alternate, fast and efficient intraoperative method for evaluation of tumor resection margins.

Development of highly reliable SERS-active photonic crystal fiber probe and its application in the detection of ovarian cancer biomarker in cyst fluid.

Conventionally Surface-enhanced Raman spectroscopy (SERS) is realized by adsorbing analytes onto nano-roughened planar substrate coated with noble metals (silver or gold) or their colloidal nanoparticles (NPs). Nanoscale irregularities in such substrates/NPs could lead to SERS sensors with poor reproducibility and repeatability. Herein, we demonstrate a suspended core photonic crystal fiber (PCF) based SERS sensor with extremely high reproducibility and repeatability in measurement with a relative SD of only 1.5% and 4.6%, respectively, which makes it more reliable than any existing SERS sensor platforms. In addition, our platform could improve the detection sensitivity owing to the increased interaction area between the guided light and the analyte, which is incorporated into the holes that runs along the length of the PCF. Numerical calculation established the significance of the interplay between light coupling efficiency and evanescent field distribution, which could eventually determine the sensitivity and reliability of the developed SERS active-PCF sensor. As a proof of concept, using this sensor, we demonstrated the detection of haptoglobin, a biomarker for ovarian cancer, contained within the ovarian cyst fluid, which facilitated in differentiating the stages of cancer. We envision that with necessary refinements, this platform could potentially be translated as a next-generation highly sensitive SERS-active opto-fluidic biopsy needle for the detection of biomarkers in body fluids.

Ultrasound Guided Optoacoustic Tomography in Assessment of Tumor Margins for Lumpectomies.

PURPOSE: To determine the accuracy of a handheld ultrasound-guided optoacoustic tomography (US-OT) probe developed for human deep-tissue imaging in ex vivo assessment of tumor margins postlumpectomy. METHODS: A custom-built two-dimensional (2D) US-OT-handheld probe was used to scan 15 lumpectomy breast specimens. Optoacoustic signals acquired at multiple wavelengths between 700 and 1100 nm were reconstructed using model linear algorithm, followed by spectral unmixing for lipid and deoxyhemoglobin (Hb). Distribution maps of lipid and Hb on the anterior, posterior, superior, inferior, medial, and lateral margins of the specimens were inspected for margin involvement, and results were correlated with histopathologic findings. The agreement in tumor margin assessment between US-OT and histopathology was determined using the Bland-Altman plot. Accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of margin assessment using US-OT were calculated. RESULTS: Ninety margins (6 × 15 specimens) were assessed. The US-OT probe resolved blood vessels and lipid up to a depth of 6 mm. Negative and positive margins were discriminated by marked differences in the distribution patterns of lipid and Hb. US-OT assessments were concordant with histopathologic findings in 87 of 89 margins assessed (one margin was uninterpretable and excluded), with diagnostic accuracy of 97.9% (kappa = 0.79). The sensitivity, specificity, PPV, and NPV were 100% (4/4), 97.6% (83/85), 66.7% (4/6), and 100% (83/83), respectively. CONCLUSION: US-OT was capable of providing distribution maps of lipid and Hb in lumpectomy specimens that predicted tumor margins with high sensitivity and specificity, making it a potential tool for intraoperative tumor margin assessment.

Surface-enhanced Raman scattering-active photonic crystal fiber probe: Towards next generation liquid biopsy sensor with ultra high sensitivity.

The tremendous enhancement factors that surface-enhanced Raman scattering (SERS) possesses coupled with the flexibility of photonic crystal fibers (PCFs) pave the way to a new generation of ultrasensitive biosensors. Thanks to the unique structure of PCFs, which allows direct incorporation of an analyte into the axially aligned air channels, interaction between the analyte and excitation light could be increased many folds leading to flexible, reliable and sensitive probes that can be used in preclinical or clinical biosensing. SERS-active PCF probes provide unique opportunity to develop an opto-fluidic liquid biopsy needle sensor that enables one-step integrated sample collection and testing for disease diagnosis. Specificity being a key parameter to biosensors, the PCF inside the biopsy needle could be functionalized with targeting moieties to detect specific biomarkers. In this review article, we present some of the most promising recent biosensors based on PCFs including hollow-core PCFs, suspended-core PCFs and side-channel PCFs. We provide a wide range of applications of such platform using Raman spectroscopy, label free SERS or labeled SERS detection and analyze some of the main challenges to be addressed for translating it to a clinically viable next generation sensitive biopsy needle sensing probe.

SERS-based detection of haptoglobin in ovarian cyst fluid as a point-of-care diagnostic assay for epithelial ovarian cancer.

PURPOSE: To evaluate haptoglobin (Hp) in ovarian cyst fluid as a diagnostic biomarker for epithelial ovarian cancers (EOCs) using surface-enhanced Raman spectroscopy (SERS)-based in vitro diagnostic assay for use in an intraoperative setting. METHODS: SERS-based method was used to detect and quantify Hp in archived ovarian cyst fluids collected from suspicious ovarian cysts and differentiate benign tumors from EOCs. The diagnostic performance of SERS-based assay was verified against the histopathology conclusions and compared with the results of CA125 test and frozen sections. RESULTS: Hp concentration present in the clinical cyst fluid measured by SERS was normalized to 3.3 mg/mL of standard Hp. Normalized mean values for patients with benign cysts were 0.65 (n=57) and malignant cysts were 1.85 (n=54), demonstrating a significantly (P<0.01) higher Hp in malignant samples. Verified against histology, Hp measurements using SERS had a sensitivity of 94% and specificity of 91%. Receiver operating characteristic curve analysis of SERS-based Hp measurements resulted in area under the curve of 0.966±0.03, establishing the robustness of the method. CA125 test on the same set of patients had a sensitivity of 85% and specificity of 90%, while frozen section analysis on 65 samples had 100% sensitivity and specificity. CONCLUSION: With a total execution time of <10 minutes and consistent performance across different stages of cancer, the SERS-based Hp detection assay can serve as a promising intra-operative EOC diagnostic test.