Publisher’s Note: This paper, originally published on 18 April 2017, was withdrawn by author's request and also because the paper was not presented at the conference.
Protein phosphorylation controls fundamental biological processes. Dysregulation of protein kinase is associated with a series of human diseases including cancer. Protein kinase A (PKA) activity has been reported to serve as a potential prognostic marker for cancer. To this end, we developed a non-radioactive, rapid, cheap and robust scheme based on surface-enhanced Raman spectroscopy (SERS) for label-free detection of PKA phosphorylation using gold nanostars (AuNS) functionalized with BSA-kemptide. While bovine serum albumin (BSA) proteins stabilized the AuNS, kemptide, which is a high affinity substrate peptide specific for PKA, were phosphorylated in vitro to generate Raman signals that were identified by performing principal component analysis (PCA) on the acquired SERS spectra.
The ever-increasing spread and emergence of antibiotic resistance poses a serious threat to global public health. With the existence of Carbapenem-resistant Enterobacteriaceae (CRE) produced by the Klebsiella Pneumoniae bacteria, it renders the use of carbapenems, the last-resort class of β-lactam antibiotics, useless against combating against bacterial infections. Such infections reduce the ability to treat complex infections due to the lack of antibiotic options for treatment, leading to CRE-associated mortalities. Current methods of detection, like CarbaNP test and Modified Hodge’s Test, have significant limitations in that the time taken for detection of carbapenemase activity ranges between hours to days, and are non-specific in detecting the specific phenotype, making it challenging to isolate patients rapidly and to devise appropriate treatment for infected patients. We propose a methodology by utilising Surface Enhanced Raman Spectroscopy (SERS) to study bacterial β-lactamase activity. This is done via the use of gold nanostars (AuNS), which have reported excellent SERS properties, conjugated with a β-lactam antibiotic ceftriaxole, as a proof-of-concept study to analyse the changes in the SERS spectra associated with cleavage of the β-lactam ring upon interaction with the New Delhi Metalloproteinase (NDM)- producing Escherichia coli (Class B β-lactamase). We are able to obtain detection of carbapenemase activity within 25 minutes, with the associated changes in SERS spectra being diminishing of SERS peaks at 1358cm-1 and 1495cm-1. This project can be further extended to study the activity of other classes of β-lactamases and other β-lactam antibiotics to improve this state of technology for potential adoption by healthcare institutions.
The control of image contrast is essential toward optimizing a contrast enhancement procedure in optical coherence tomography (OCT). In this study, the in vivo control of optical contrast in a mouse tumor model with gold nanoshells as a contrast agent is examined. Gold nanoshells are administered into mice, with the injected dosage and particle surface parameters varied and its concentration in the tumor under each condition is determined using a noninvasive theoretical OCT modeling technique. The results show that too high a concentration of gold nanoshells in the tumor only enhances the OCT signal near the tissue surface, while significantly attenuating the signal deeper into the tissue. With an appropriate dosage, IV delivery of gold nanoshells allows a moderate concentration of 6.2×109 particles/ml in tumor to achieve a good OCT signal enhancement with minimal signal attenuation with depth. An increase in the IV dosage of gold nanoshells reveals a corresponding nonlinear increase in their tumor concentration, as well as a nonlinear reduction in the fractional concentration of injected gold nanoshells. Furthermore, this fractional concentration is improved with the use of antiepodermal growth factor receptor (EGFR) surface functionalization, which also reduces the time required for tumor delivery from 6 to 2 h.
Our aim is to apply image analysis on photosensitizer fluorescence and compare the relationship between histopathology and endoscopic fluorescence imaging. The correlation between hypericin fluorescence and histopathology of diseased tissue was explored in a clinical study involving 58 fluorescence cystoscopic images from 23 patients. Based on quantification of fluorescence colorimetric parameters extracted from the image analysis, diagnostic functions were developed to pathologically classify the bladder cancer. Our preliminary results show that the differences in fluorescence intensity ratios among the three different grades of bladder cancer are statistically significant. The results also show a decrease in macroscopic fluorescence intensity that correlated with higher cancer grades. By combining both the red-to-green and red-to-blue fluorescence intensity ratios into a 2-D scatter plot and defining diagnostic linear discrimination functions on the data points, this technique is able to yield an average sensitivity and specificity of around 68.6% and 86.1%, respectively, for pathological cancer grading of the three different grades of bladder cancer in our study. We conclude that our proposed approach in applying colorimetric intensity ratio analysis on hypericin fluorescence shows potential to optically grade bladder cancer in situ.
The application of gold nanoparticles as a contrast agent in optical bioimaging is well appreciated, but limited to a
narrow excitation range due to its rather invariable optical resonance typically at 520 nm. Compared to gold
nanoparticles, the optical response of gold nanoshells can be tuned to match the higher excitation wavelength of many
promising clinical reflectance-based imaging modalities such as the optical coherence tomography (OCT). In this study,
we demonstrate the tunability of gold nanoshells to improve the optical contrast of backscattering signal under confocal
reflectance microscopy and OCT. The gold nanoshells were synthesized and conjugated to antibodies for in vitro
demonstration of their selective optical contrast in cancer cells over normal cells under the confocal reflectance
microscopy. The OCT signals from these gold nanoshells were compared to that from bare silica cores and intrinsic
tissue scattering using 1% Intralipid. We have shown that gold nanoshells are able to elicit an optical contrast to
discriminate between cancerous and normal cells under the confocal reflectance microscopy based on differences in
molecular markers expression. Compared to bare silica core, the presence of the gold shell is able to effect a higher
backscattered OCT signal with an apparent contrast over 1% Intralipid. This contrast can be made to be dependent on
the molecular marker expression with antibody specificity.
Estimation of the tissue optical characteristics using optical coherence tomography (OCT) requires good modeling.
Present modeling of the system includes effects such as scattering of light in tissues. However, absorption effects were
often neglected in the model. They may be significant depending on the tissue type and the wavelength of the light
source. We present a study where the effects of absorption in light propagation in biological tissue were examined in the
theoretical modeling of OCT based on the single-scattering model. OCT M-scans were performed on liquid tissue
phantoms at 1% concentration. In order to mimic the effects of absorption, India ink was added to the solution. Different
concentrations of Indian ink were used to vary the absorption coefficient in the tissue phantoms. Estimation of the
absorption, scattering coefficients from the OCT signal were obtained. Substantial reduction in the slope of the
logarithmic OCT signal was observed when India ink was introduced to the liquid tissue phantoms. The results suggest
that the effects of the absorption clearly affected the estimation of the overall extinction coefficient. In order to improve
the accuracy of estimation of these characteristics, absorption effects should be taken into account.
Advanced optical technologies for in vivo imaging e.g. OCT and confocal reflectance endomicroscopy while being able to image stromal morphology, are unable to image biomolecular changes associated with carcinogenesis. Furthermore, the contrast between neoplastic and normal tissues from such advanced optical technologies is often too low to be of any clinical value. Due to their favorable optical properties including their ability to resonantly scatter light at surface plasmon resonance to present potentially good contrast for reflectance-mode imaging, we aim to develop gold nanoparticles as optical contrast agents coupled with these optical imaging systems to perform cancer targeting bioimaging for early diagnosis of epithelial carcinoma. In this study, 20 nm gold nanoparticles were synthesized and conjugated with anti-EGFR (Epidermal Growth Factor Receptor). EGFR is a cell surface receptor biomarker that is highly expressed in majority of epithelial cancer compared to normal cells. The resulting anti-EGFR conjugated gold nanoparticles were allowed to interact with the nasopharyngeal carcinoma CNE2 cells in vitro. The exact localization of the gold bioconjugates on the cell surface EGFR receptors was investigated using confocal immunofluorescence microscopy. We have demonstrated that the binding and localization of the gold bioconjugates on the cell surface increased the reflectance and scattering properties of the CNE2 cells and provide good optical contrast for the cancer cells under confocal reflectance microscopy. Thus our study has demonstrated the potential of gold nanoparticles to
target and illuminate cancer cells for bioimaging.
Photodynamic diagnosis of bladder carcinoma based on hypericin fluorescence cystoscopy has shown to have a higher degree of sensitivity for the detection of flat bladder carcinoma compared to white light cystoscopy. The potential of the photosensitizer hypericin-induced fluorescence in performing non-invasive optical biopsy to grade bladder cancer in vivo using fluorescence cystoscopic image analysis without surgical resection for tissue biopsy is investigated in this study. The correlation between tissue fluorescence and histopathology of diseased tissue was explored and a diagnostic algorithm based on fluorescence image analysis was developed to classify the bladder cancer without surgical resection for tissue biopsy. Preliminary results suggest a correlation between tissue fluorescence and bladder cancer grade. By combining both the red-to-blue and red-to-green intensity ratios into a 2D scatter plot yields an average sensitivity and specificity of around 70% and 85% respectively for pathological cancer grading of the three different grades of bladder cancer. Therefore, the diagnostic algorithm based on colorimetric intensity ratio analysis of hypericin fluorescence
cystoscopic images developed in this preliminary study shows promising potential to optically diagnose and grade bladder cancer in vivo.
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