Refinement of an open-microcavity optical biosensor for bacterial endotoxin test.

The Limulus Amebocyte Lysate (LAL) test is an in vitro assay widely used in the pharmaceutical and biotechnology industries to detect bacterial endotoxins. Endotoxin is a structural component of the cell wall of Gram-negative bacteria, which has serious pathogenic effects in the body and may cause dysfunction of multiple organ systems and increased risk of mortality. To address the growing need for LAL assays due to the increased demand from drug and vaccine manufacturers, we have developed a new LAL assay approach. Our detection mechanism is different and improved from those currently used in the industry, leading to increased test sensitivity and reduced assay time. Our study utilizes an open-microcavity photonic-crystal biosensor to quantify endotoxin concentrations. It has demonstrated an improved LAL assay sensitivity by 10 fold compared to the commercial standard methods and reduced the time needed for the assay by more than half. In addition, this approach requires as little as 5 µL of LAL reagent per test, thereby decreasing costs and conserving horseshoe crabs. The results reported in this paper indicate the possibility of using the photonic-crystal biosensor based approach for significant enhancements of endotoxin testing.

Prostate Cancer Epithelial Mesenchymal Transition by TGF-ß1 exposure monitored with a Photonic Crystal Biosensor.

During prostate cancer progression, cancerous epithelial cells can undergo epithelial-mesenchymal transition (EMT). EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. It is associated with biochemical changes such as epithelial cell markers E-cadherin and occludins being down-regulated, and mesenchymal markers vimentin and N-cadherin being upregulated. These changes in protein expression, specifically in the cell membrane, may be monitored via biophysical principles, such as changes in the refractive index (RI) of the cell membrane. In our previous research, we demonstrated the feasibility of using cellular RI as a unique contrast parameter to accomplish label-free detection of prostate cancer cells. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free biosensing system, which allows for ultra-sensitive quantification of the changes in cellular RI due to EMT. We induced prostate cancer cells to undergo EMT by exposing these cells to soluble Transforming Growth Factor Beta 1 (TGF-ß1). The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced cancer cells. Our study shows promising clinical potential in utilizing the PC-TIR biosensing system not only to detect prostate cancer cells, but also to evaluate changes in prostate cancer cells due to EMT.

Cellular Refractive Index Comparison of Various Prostate Cancer and Noncancerous Cell Lines via Photonic-Crystal Biosensor.

The current clinical standard for mass screening of prostate cancer are prostate-specific antigen (PSA) biomarker assays. Unfortunately, the low specificity of PSA's bioassays to prostate cancer leads to high false-positive rates, as such there is an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we have successfully demonstrated, with the use of our Photonic-Crystal based biosensor in a Total-Internal-Reflection (PC-TIR) configuration, detection of prostate cancer (PC-3) cells against benign prostate hyperplasia (BPH-1) cells. The PC-TIR biosensor achieved detection of individual prostate cancer cells utilizing cellular refractive index (RI) as the only contrast parameter. To further study this methodology in vitro, we report a comprehensive study of the cellular RI's of various prostate cancer and noncancerous cell lines (i.e. RWPE-1, BPH-1, PC-3, DU-145, and LNCaP) via reflectance spectroscopy and single-cell RI imaging utilizing the PC-TIR biosensor. Our study shows promising clinical potential in utilizing the PC-TIR biosensor system for the detection of prostate cancer against noncancerous prostate epithelial cells.

Segmentation of breast ultrasound images based on active contours using neutrosophic theory.

PURPOSE: Ultrasound imaging is an effective approach for diagnosing breast cancer, but it is highly operator-dependent. Recent advances in computer-aided diagnosis have suggested that it can assist physicians in diagnosis. Definition of the region of interest before computer analysis is still needed. Since manual outlining of the tumor contour is tedious and time-consuming for a physician, developing an automatic segmentation method is important for clinical application. METHODS: The present paper represents a novel method to segment breast ultrasound images. It utilizes a combination of region-based active contour and neutrosophic theory to overcome the natural properties of ultrasound images including speckle noise and tissue-related textures. First, due to inherent speckle noise and low contrast of these images, we have utilized a non-local means filter and fuzzy logic method for denoising and image enhancement, respectively. This paper presents an improved weighted region-scalable active contour to segment breast ultrasound images using a new feature derived from neutrosophic theory. RESULTS: This method has been applied to 36 breast ultrasound images. It generates true-positive and false-positive results, and similarity of 95%, 6%, and 90%, respectively. CONCLUSION: The purposed method indicates clear advantages over other conventional methods of active contour segmentation, i.e., region-scalable fitting energy and weighted region-scalable fitting energy.

Label-free in vitro prostate cancer cell detection via photonic-crystal biosensor.

Prostate-specific antigen (PSA) biomarker assays are the current clinical method for mass screening of prostate cancer. However, high false-positive rates are often reported due to PSA's low specificity, leading to an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we demonstrated the feasibility of using cellular refractive indices (RI) as a unique contrast parameter to accomplish label-free detection of prostate cancer cells via variance testing, but were unable to determine if a specific cell was cancerous or noncancerous. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free imaging system, which allows for the detection of individual prostate cancer cells utilizing cellular RI as the only contrast parameter. Noncancerous prostate (BPH-1) cells and prostate cancer (PC-3) cells were mixed at varied ratios and measured concurrently. Additionally, we isolated and induced PC-3 cells to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors such as TGF-01. The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced PC-3 cells as well as BPH-1 cells. EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. Our study shows promising clinical potential in utilizing the PC-TIR biosensor imaging system to not only detect prostate cancer cells, but also evaluate prostate cancer progression.

Study of the Interactions of Fusarium virguliforme Toxin FvTox1 with Synthetic Peptides by Molecular Simulations and a Label-Free Biosensor.

Fusarium virguliforme is a soil borne pathogen that causes sudden death syndrome (SDS) in soybean plants. This pathogenic disease may result in severe soybean yield suppression and can cause serious economic harm. It has been shown that the FvTox1 toxin produced by the pathogen may be the root cause of foliar SDS. Anti-FvTox1 single-chain variable fragment antibody expressed in transgenic soybean plants was shown to neutralize the FvTox1 toxin involved in foliar SDS development. Here, we have investigated the binding affinities of FvTox1 with four FvTox1-interacting peptides of 7 to 12 amino acids identified from phage display libraries using both bioinformatics-based molecular simulations and label-free bioassays with a unique photonic crystal biosensor. Results from the molecular simulations have predicted the interaction energies and 3-dimensional (3D) structures of FvTox1 and FvTox1-interacting peptide complexes. Our label-free binding assays have further provided the interaction strength of FvTox1 with four different FvTox1-interacting peptides and experimentally confirmed the simulation results obtained from bioinformatics-based molecular calculations.

Optical fiber-based in vivo quantification of growth factor receptors.

BACKGROUND: Growth factor receptors such as epidermal growth factor receptor 1 and human epidermal growth receptor 2 (HER2) are overexpressed in certain cancer cells. Antibodies against these receptors (eg. cetuximab and transtuzumab [Herceptin]) have shown therapeutic value in cancer treatment. The existing methods for the quantification of these receptors in tumors involve immunohistochemistry or DNA quantification, both in extracted tissue samples. The goal of the study was to evaluate whether an optical fiber-based technique can be used to quantify the expression of multiple growth factor receptors simultaneously. METHODS: The authors examined HER2 expression using the monoclonal antibody trastuzumab as a targeting ligand to test their system. They conjugated trastuzumab to 2 different Alexa Fluor dyes with different excitation and emission wavelengths. Two of the dye conjugates were subsequently injected intravenously into mice bearing HER2-expressing subcutaneous tumors. An optical fiber was then inserted into the tumor through a 30-gauge needle, and using a single laser beam as the excitation source, the fluorescence emitted by the 2 conjugates was identified and quantified by 2-photon optical fiber fluorescence. RESULTS: The 2 conjugates bound to the HER2-expressing tumor competitively in a receptor-specific fashion, but they failed to bind to a similar cell tumor that did not express HER2. The concentration of the conjugate present in the tumor as determined by 2-photon optical fiber fluorescence was shown to serve as an index of the HER2 expression levels. CONCLUSIONS: These studies offer a minimally invasive technique for the quantification of tumor receptors simultaneously.

Enhanced optical breakdown in KB cells labeled with folate-targeted silver-dendrimer composite nanodevices.

Enhanced optical breakdown of KB tumor cells folate-targeted with silver-dendrimer composite nanodevices (CNDs) is described. CNDs [(Ag(0))(25)-PAMAM_E5.(NH(2))(42)(NGly)(74)(NFA)(2.7)] were fabricated by reactive encapsulation, using a biocompatible template of dendrimer-folic acid (FA) conjugates. Preferential uptake of the folate-targeted CNDs (of various treatment concentrations and surface functionality) by KB cells was visualized with confocal microscopy and transmission electron microscopy. Intracellular laser-induced optical breakdown threshold and dynamics were detected and characterized by high-frequency ultrasonic monitoring of resulting transient bubble events. When irradiated with a near-infrared, femtosecond laser, the CND-targeted KB cells acted as well-confined activators of laser energy, enhancing nonlinear energy absorption, exhibiting a significant reduction in breakdown threshold and thus selectively promoting intracellular laser-induced optical breakdown. FROM THE CLINICAL EDITOR: This study presents a novel method to selectively destroy cancer cells by combining biochemical targeting with topical laser irradiation. A human epidermoid cancer cell line was targeted with folated silver-dendrimer composite nanodevices and the labeled cancer cells were subsequently destroyed by the microbubbles generated due the enhanced energy uptake of the silver nanoparticles from the laser irradiation, as compared to unlabeled cells.

Fiber-optic multiphoton flow cytometry in whole blood and in vivo.

Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.

Two-photon in vivo flow cytometry using a fiber probe.

We have demonstrated the use of a double-clad fiber probe to conduct two-photon excited flow cytometry in vitro and in vivo. We conducted two-channel detection to measure fluorescence at two distinct wavelengths simultaneously. Because the scattering and absorption problems from whole blood were circumvented by the fiber probe, the detected signal strength from the cells were found to be similar in PBS and in whole blood. We achieved the same detection efficiency of the membrane-binding lipophilic dye DiD labeled cells in PBS and in whole blood. High detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was demonstrated. DiD-labeled untransfected and GFP-transfected cells were injected into live mice and the circulation dynamics of the externally injected cells were monitored. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed in whole blood.

Investigation of tumor cell targeting of a dendrimer nanoparticle using a double-clad optical fiber probe.

Fluorescence quantification in tissues using conventional techniques can be difficult due to the absorption and scattering of light in tissues. Our previous studies have shown that a single-mode optical fiber (SMF)-based, two-photon optical fiber fluorescence (TPOFF) probe could be effective as a minimally invasive, real-time technique for quantifying fluorescence in solid tumors. We report improved results with this technique using a solid, double-clad optical fiber (DCF). The DCF can maintain a high excitation rate by propagating ultrashort laser pulses down an inner single-mode core, while demonstrating improved collection efficiency by using a high-numerical aperture multimode outer core confined with a second clad. We have compared the TPOFF detection efficiency of the DCF versus the SMF with standard solutions of the generation 5 poly(amidoamine) dendrimer (G5) nanoparticles G5-6TAMRA (G5-6T) and G5-6TAMRA-folic acid (G5-6T-FA). The DCF probe showed three- to five-fold increases in the detection efficiency of these conjugates, in comparison to the SMF. We also demonstrate the applicability of the DCF to quantify the targeted uptake of G5-6T-FA in mouse tumors expressing the FA receptor. These results indicate that the TPOFF technique using the DCF probe is an appropriate tool to quantify low nanomolar concentrations of targeted fluorescent probes from deep tissue.

Dendrimer-epidermal growth factor conjugate displays superagonist activity.

Binding of ligands on to epidermal growth factor receptor (EGFR) can stimulate cell growth; therefore, any application employing EGF as a targeting ligand for a "drug carrier" must evaluate the effect of the conjugate on cell growth. We report the synthesis and in vitro biological activity of EGF molecules coupled to a fluorescein-labeled polyamidoamine dendrimer. The conjugate bound and internalized into several EGFR-expressing cell lines in a receptor-specific fashion. The conjugate effectively induced EGFR phosphorylation and acted as a superagonist by stimulating cell growth to a greater degree than free EGF. Concomitant administration of the chemotherapeutic drug methotrexate completely inhibited cell growth to a degree similar to its effect in the absence of the conjugate. Thus, dendrimer-EGF conjugates serve as EGFR superagonists, but this activity can be overcome by chemotherapeutic drugs. The agonist activity of these materials must be taken into consideration when using EGF conjugates for imaging applications.

In Vivo Monitoring of Multiple Circulating Cell Populations Using Two-photon Flow Cytometry.

To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes.

Acoustic estimation of thermal distribution in the vicinity of femtosecond laser-induced optical breakdown.

Laser-induced optical breakdown (LIOB), or photo-disruption, can generate individual microbubbles in tissues for biomedical applications. We have previously developed a co-localized high-frequency ultrasound system to detect and characterize these laser-induced microbubbles. Because ultrasound speed varies with temperature, this system can also be used to directly estimate thermal effects in the vicinity of photodisruption. In this study, individual bubbles (sizes 60-100 microm) were created at the bottom of a water tank using a 793-nm, 100-fs Ti:Sapphire laser pulsed at 250 kHz. During and after breakdown, pulse-echoes from the tank bottom in the region surrounding a bubble were recorded with a single-element 85-MHz ultrasonic transducer, and temperature-dependent pulse-echo displacements were calculated using phase-sensitive correlation tracking. These displacements were then fit to a finite-element heat transfer model to estimate the effective thermal distribution. Estimates were calculated for laser exposure times ranging from 6.25 to 312.5 ms (1600 to 78 000 laser pulses), at 1.5 and 4 J/cm2 fluences. Results suggest a minimal temperature increase (<1 degrees C) within 100 microm of a bubble created with <1600 laser pulses at 1.5 J/cm2 fluence. This implies that LIOB can be controlled to be thermally noninvasive in the bubble vicinity.

Optical and acoustic detection of laser-generated microbubbles in single cells.

Acoustically monitored laser-induced optical breakdown (LIOB) has potential as an important tool to diagnose and treat living cells. Laser-induced intracellular microbubbles are readily detectable using high-frequency ultrasound, and LIOB can be controlled to operate within two distinct regimes. In the nondestructive regime, a single, short-lived bubble can be generated within a cell, without affecting its immediate viability. In the destructive regime, the induced photodisruption quickly can kill a targeted cell. To generate and monitor this range of bioeffects in real time, we have developed a system integrating an ultrafast laser source with optical and acoustic microscopy. Experiments were performed on monolayers of Chinese hamster ovary (CHO) cells. A 793 nm, 100 fs laser pulsed at 3.8 kHz was tightly focused within each cell to produce the photodisruption, and a 50 MHz ultrasonic transducer monitored the resultant bubble via continuous pulse-echo recordings. Photodisruption was also observed using bright field microscopy, and cell viability was assessed following laser exposure with a trypan blue assay. By controlling laser pulse fluence and exposure duration, either nondestructive or destructive LIOB could be produced. The intracellular position of the laser focus was also varied to demonstrate that cell viability was affected by the specific location of material breakdown.

In vitro targeting of synthesized antibody-conjugated dendrimer nanoparticles.

This study reports the synthesis and in vitro biological properties of dendrimer-antibody conjugates. The polyamidoamine dendrimer platform was conjugated to fluorescein isothiocyanate as a means to analyze cell binding and internalization. Two different antibodies, 60bca and J591, which bind to CD14 and prostate-specific membrane antigen (PSMA), respectively, were used as model targeting molecules. The binding of the antibody-conjugated dendrimers to antigen-expressing cells was evaluated by flow cytometry, confocal microscopy, and a new two-photon-based optical fiber fluorescence detection system. The conjugates specifically bound to the antigen-expressing cells in a time- and dose-dependent fashion, with affinity similar to that of the free antibody. Confocal microscopic analysis suggested at least some cellular internalization of the dendrimer conjugate. Dendrimer-antibody conjugates are a suitable platform for targeted molecule delivery into antigen-expressing cells.

Detection and analysis of tumor fluorescence using a two-photon optical fiber probe.

The utility of a two-photon optical fiber fluorescence probe (TPOFF) for sensing and quantifying tumor fluorescent signals was tested in vivo. Xenograft tumors were developed in athymic mice using MCA207 cells expressing green fluorescent protein (GFP). The TPOFF probe was able to detect ex vivo fluorescence from excised tumors containing as little as 0.3% GFP-expressing cells. TPOFF results were similar to both flow-cytometric analysis of tumor cells after isolation and suspension, and fluorescence determined by microscope images of cryosectioned tumors. TPOFF was then used to measure GFP fluorescence from tumors in live mice. The fiber probe detected fluorescently-labeled Herceptin antibody targeted to HER2-expressing tumors in severe combined immunodeficient mice. Dendrimer nanoparticles targeted by folic acid and having 6-TAMRA as a fluorescent probe were also used to label KB cell tumors in vivo. The fiber probe documented a fourfold increase in tumor fluorescence in animals that received the targeted dendrimer. These results suggest TPOFF can be used as a minimally invasive system for identifying tumor markers and monitoring drug therapy.

Biosensing based on two-photon fluorescence measurements through optical fibers.

We have performed two-photon fluorescence detection in a new scheme in which femtosecond laser pulses were delivered thorugh an optical fiber for nonlinear excitation and the emitted fluorescence was collected through the same fiber. Single-mode fibers were determined to give higher detection efficiency than multimode fibers, consistent with theoretical considerations. The utility of fiber-optic sensing based on two-photon fluorescence detection was proved by an experiment that measured the uptake of a targeted drug delivery agent into cultured cancer cells.