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.

Identification of mycolic acid forms using surface-enhanced Raman scattering as a fast detection method for tuberculosis.

BACKGROUND: Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, based on the WHO Global Tuberculosis Report in 2017. TB causes massive health care burdens in many parts of the world, specifically in the resource constrained developing world. Most deaths from TB could be prevented with cost effective early diagnosis and appropriate treatment. PURPOSE: Conventional TB detection methods are either too slow as it takes a few weeks for diagnosis or they lack the specificity and accuracy. Thus the objective of this study was to develop a fast and efficient detection for TB using surface enhanced Raman scattering (SERS) technique. METHODS: SERS spectra for different forms of mycolic acids (MAs) that are both synthetic origin and actual extracts from the mycobacteria species were obtained by label-free direct detection mode. Similarly, we collected SERS spectra for γ-irradiated whole bacteria (WB). Measurements were done using silver (Ag) coated silicon nanopillar (Ag SNP) as SERS substrate. RESULTS: We report the SERS based detection of MA, which is a biomarker for mycobacteria species including Mycobacterium tuberculosis. For the first time, we also establish the SERS spectral characterization of the three major forms of MA - αMA, methoxy-MA, and keto-MA, in bacterial extracts and also in γ-irradiated WB. We validated our findings by mass spectrometry. SERS detection of these three forms of MA could be useful in differentiating pathogenic and nonpathogenic Mycobacterium spp. CONCLUSIONS: We have demonstrated the direct detection of three major forms of MA - αMA, methoxy-MA, and keto-MA, in two different types of MA extracts from MTB bacteria, namely delipidated MA and undelipidated MA and finally in γ-irradiated WB. In the near future, this study could pave the way for a fast and efficient detection method for TB, which is of high clinical significance.

Noninvasive real-time characterization of non-melanoma skin cancers with handheld optoacoustic probes.

Currently, imaging technologies that enable dermsurgeons to visualize non-melanoma skin cancers (NMSC) in vivo preoperatively are lacking, resulting in excessive or incomplete removal. Multispectral optoacoustic tomography (MSOT) is a volumetric imaging tool to differentiate tissue chromophores and exogenous contrast agents, based on differences in their spectral signatures and used for high-resolution imaging of functional and molecular contrast at centimeter scale depth. We performed MSOT imaging with two- and three-dimensional handheld scanners on 21 Asian patients with NMSC. The tumors and their oxygenation parameters could be distinguished from normal skin endogenously. The lesion dimensions and depths were extracted from the spectral melanin component with three-dimensional spatial resolution up to 80 µm. The intraclass correlation coefficient correlating tumor dimension measurements between MSOT and ex vivo histology of excised tumors, showed good correlation. Real-time 3D imaging was found to provide information on lesion morphology and its underlying neovasculature, indicators of the tumor's aggressiveness.

Enhancement of early cervical cancer diagnosis with epithelial layer analysis of fluorescence lifetime images.

This work reports the use of layer analysis to aid the fluorescence lifetime diagnosis of cervical intraepithelial neoplasia (CIN) from H&E stained cervical tissue sections. The mean and standard deviation of lifetimes in single region of interest (ROI) of cervical epithelium were previously shown to correlate to the gold standard histopathological classification of early cervical cancer. These previously defined single ROIs were evenly divided into layers for analysis. A 10-layer model revealed a steady increase in fluorescence lifetime from the inner to the outer epithelial layers of healthy tissue sections, suggesting a close association with cellular maturity. The shorter lifetime and minimal lifetime increase towards the epithelial surface of CIN-affected regions are in good agreement with the absence of cellular maturation in CIN. Mean layer lifetimes in the top-half cervical epithelium were used as feature vectors for extreme learning machine (ELM) classifier discriminations. It was found that the proposed layer analysis technique greatly improves the sensitivity and specificity to 94.6% and 84.3%, respectively, which can better supplement the traditional gold standard cervical histopathological examinations.

Quantitative diagnosis of cervical neoplasia using fluorescence lifetime imaging on haematoxylin and eosin stained tissue sections.

The use of conventional fluorescence microscopy for characterizing tissue pathological states is limited by overlapping spectra and the dependence on excitation power and fluorophore concentration. Fluorescence lifetime imaging microscopy (FLIM) can overcome these limitations due to its insensitivity to fluorophore concentration, excitation power and spectral similarity. This study investigates the diagnosis of early cervical cancer using FLIM and a neural network extreme learning machine classifier. A concurrently high sensitivity and specificity of 92.8% and 80.2%, respectively, were achieved. The results suggest that the proposed technique can be used to supplement the traditional histopathological examination of early cervical cancer.

Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber.

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-µg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ∼100 pg in a sample volume of ∼10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.

New frontier in hypericin-mediated diagnosis of cancer with current optical technologies.

Photosensitizers (PSs) have shown great potentials as molecular contrast agents in photodynamic diagnosis (PDD) of cancer. While the diagnostic values of PSs have been proven previously, little efforts have been put into developing optical imaging and diagnostic algorithms. In this article, we review the recent development of optical probes that have been used in conjunction with a potent PS, hypericin (HY). Various fluorescence techniques such as laser confocal microscopy, fluorescence urine cytology, endoscopy and endomicroscopy are covered. We will also discuss about image processing and classification approaches employed for accurate PDD. We anticipate that continual efforts in these developments could lead to an objective PDD and complete surgical clearance of tumors. Recent advancements in nanotechnology have also opened new horizons for PSs. The use of biocompatible gold nanoparticles as carrier for enhanced targeted delivery of HY has been attained. In addition, plasmonic properties of nanoparticles were harnessed to induce localized hyperthermia and to manage the release of PS molecules, enabling a better therapeutic outcome of a combined photodynamic and photothermal therapy. Finally, we discuss how nanoparticles can be used as contrast agents for other optical techniques such as optical coherence tomography and surface-enhanced Raman scattering imaging.

Nanosphere templated metallic grating assisted enhanced fluorescence.

In this paper, enhanced fluorescence from a silver film coated nanosphere templated grating is presented. Initially, numerical simulation was performed to determine the plasmon resonance wavelength by varying the thickness of the silver film on top of a monolayer of 400 nm nanospheres. The simulation results are verified experimentally and tested for enhancing fluorescence from fluorescein isothiocyanate whose excitation wavelength closely matches with the plasmon resonance wavelength of the substrate with 100 nm silver film over nanosphere. The 12 times enhancement in the intensity is attributed to the local field enhancement in addition to the excitation of surface plasmon polaritons along the surface.

Clinical SERS: are we there yet?

Surface Enhanced Raman Spectroscopy or SERS has witnessed many successes over the past 3 decades, owing particularly to its simplicity of use as well as its highly-multiplexing capability. This article provides an overview of SERS and its applicability in the field of bio-medicine. We will preview recent developments in SERS substrate designs, and the various sensing technologies that are based on the SERS phenomenon. An overview of the clinical applications of SERS is also included. Finally, we provide an opinion on the future trends of this unique spectroscopic technique.

Development of biocompatible SERS nanotag with increased stability by chemisorption of reporter molecule for in vivo cancer detection.

Biocompatible surface-enhanced Raman scattering (SERS) nanotag has been developed by chemisorption of novel Raman reporters on gold colloid. We modified our previously published best five reporter molecules (B2, B7, C3, C7 and C9) from triphenylmethine (TM) library using lipoic acid (LA) as a linker to covalently attach the reporters on gold colloid. Among these TM-LA molecules, B2LA showed the highest SERS signal intensity and stability over time. Further, time course SERS intensity of B2LA was compared with currently popular Raman reporter malachite green isothiocyanate (MGITC). The results demonstrated that signal intensity from B2LA was even stable over a period of one month. In vitro SERS screening was performed in cancer cell lines using B2LA containing nanotag conjugated with selective antibodies recognizing HER2 and EGFR cancer proteins. We found reasonably strong SERS signals from both HER2 and EGFR positive cells whereas no signal was measured from respective negative cells. Moreover, we successfully proved this recognition by cell imaging using fluorescein isothiocyanate (FITC) labeled antibody conjugated nanotag. Both SERS and cell-imaging study further confirmed the selective binding of antibody conjugated nanotag to cancer cells over-expressing HER2 and EGFR. In addition, as a proof of concept, in vivo SERS measurement in a mouse model was carried out to detect the nanotag-anchored cancer cells that are subcutaneously injected to the animal.

Combinatorial synthesis of a triphenylmethine library and their application in the development of surface enhanced Raman scattering (SERS) probes.

The first synthesis of a triphenylmethine (TM) library of compounds and screening of their Surface Enhanced Raman Scattering (SERS) capability was carried out to identify novel Raman reporters with high sensitivity. We identified three novel SERS reporters (B2, B7, and C7) with higher signal intensity than that of commonly used crystal violet (CV). These reporters may find potential applications in developing sensitive SERS based biosensors.

Fluorescence lifetime discrimination using expectation-maximization algorithm with joint deconvolution.

The fluorescence lifetime technique offers an effective way to resolve fluorescent components with overlapping emission spectra. The presence of multiple fluorescent components in biological compounds can hamper their discrimination. The conventional method based on the nonlinear least-squares technique is unable to consistently determine the correct number of fluorescent components in a fluorescence decay profile. This can limit the applications of the fluorescence lifetime technique in biological assays and diagnoses where more than one fluorescent component is typically encountered. We describe the use of an expectation-maximization (EM) method with joint deconvolution to estimate the fluorescence decay parameters, and the Bayesian information criterion (BIC) to accurately determine the number of fluorescent components. A comprehensive simulation and experimental study is carried out to compare the performance and accuracy of the proposed method. The results show that the EM-BIC method is able to accurately identify the correct number of fluorescent components in samples with weakly fluorescing components.

Effects of N-methyl pyrrolidone on the uptake of hypericin in human bladder carcinoma and co-staining with DAPI investigated by confocal microscopy.

Photodynamic diagnosis (PDD) using hypericin (HY), a natural photosensitizer, detects bladder cancer significantly better than white light endoscopy. However, the lipophilicity of HY complicates its administration for clinical applications. Currently, pharmaceutical preparations for HY without plasma protein are being developed. Formulations containing a biocompatible solvent, N-methyl pyrrolidone (NMP) have been shown to enhance the photodynamic therapeutic effects of HY. It was recently reported that, NMP formulations of HY were able to produce significantly higher contrast for fluorescence detection of tumors than albumin-containing HY formulations. This present work hypothesizes that NMP acts both as a solvent and penetration enhancer to improve the delivery of HY into cells by increasing the permeability of cell membranes. This paper reports the use of 3-D confocal microscopy to monitor real-time uptake of HY in human carcinoma. 3-D confocal microscopy was used to investigate the possibility of nuclear localization of HY in MGH cells. The fluorescence of HY was confirmed to be emitted from HY containing cells using spectrometry. The localization of a DNA fluorescent probe 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to confirm the possibility of colocalization of DAPI and HY. The colocalization analysis in the present study suggests that it was very unlikely that HY colocalized in the nucleus that was stained by DAPI. Fluorescein leakage tests showed that 1% NMP changes the permeability of cell membranes, and enhanced the delivery of HY into cells resulting in lower cell survival ratios. Thus, NMP was able to enhance the photodynamic therapeutic effects of HY on cancer cells.

Fluorescence detection of bladder cancer using urine cytology.

Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate in bladder cancer can be reduced by early treatment following pre-cancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimens, which is both invasive and time-consuming. In this study, ex vivo urine fluorescence cytology was investigated to offer an alternative timely and biopsy-free means for detecting bladder cancers. Sediments in patient urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cancer cells from the other cells based on the cellular size. Intensity histograms of each targeted cell and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cancer cells were observed. A diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this report. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.

Integration of optical fiber light guide, fluorescence detection system, and multichannel disposable microfluidic chip.

A combination of fluorescence detection and microfluidic technology provides promising applications in life sciences. A prototype of an integrated fluorescence detection system and optical fiber light guide on a laminate-based multichannel microfluidic chip has been developed and tested. A blue LED, plastic optical fiber, photodiode, Mylar and PMMA, and fluorescein and BSA-FITC were used as an excitation source, light coupler and guide, detector, microfluidic substrate and sample, respectively. The results show that the system is capable of detecting weak fluorescence emission from a fluorescein solution at concentration down to 0.01 ng/ml, and gives linear response. The results were also reproducible, and no cross-talk between adjacent channels was observed. The test using BSA as a model analyte demonstrates its feasibility for on-chip immunosensor applications. The performance and applications can be developed further. This prototype can be used as a platform to develop a simple and compact bio-fluorescence detection system integrated with an inexpensive and disposable multichannel microfluidic chip for biomedical devices.

Compact fluorescence detection using in-fiber microchannels-its potential for lab-on-a-chip applications.

This paper reports a compact and practical fluorescence sensor using an in-fiber microchannel. A blue LED, a multimode PMMA or silica fiber and a mini-PMT were used as an excitation source, a light guide and a fluorescence detector, respectively. Microfluidic channels of 100 microm width and 210 microm depth were fabricated in the optical fibers using a direct-write CO(2) laser system. The experimental results show that the sensor has high sensitivity, able to detect 0.005 microg L(-1) of fluorescein in the PBS solution, and the results are reproducible. The results also show that the silica fiber sensor has better sensitivity than that of the PMMA fiber sensor. This could be due to the fouling effect of the frosty layer formed at the microchannel made within the PMMA fiber. It is believed that this fiber sensor has the potential to be integrated into microfluidic chips for lab-on-a-chip applications.