Ruimin Tan

Application of Photonic Crystal Enhanced Fluorescence to Cancer Biomarker Microarrays

We report on the use of photonic crystal surfaces as a high-sensitivity platform for detection of a panel of cancer biomarkers in a protein microarray format. The photonic crystal surface is designed to provide an optical resonance at the excitation wavelength of cyanine-5 (Cy5), thus providing an increase in fluorescent intensity for Cy5-labeled analytes measured with a confocal microarray scanner, compared to a glass surface. The sandwich enzyme-linked immunosorbent assay (ELISA) is undertaken on a microarray platform to undertake a simultaneous, multiplex analysis of 24 antigens on a single chip. Our results show that the resonant excitation effect increases the signal-to-noise ratio by 3.8- to 6.6-fold, resulting in a decrease in detection limits of 6-89%, with the exact enhancement dependent upon the antibody-antigen interaction. Dose-response characterization of the photonic crystal antibody microarrays shows the capability to detect common cancer biomarkers in the <2 pg/mL concentration range within a mixed sample.

Cellular recognition and trafficking of amorphous silica nanoparticles by macrophage scavenger receptor A

Abstract The cellular uptake of engineered nanoparticles (ENPs) is known to involve active transport mechanisms, yet the biological molecules involved are poorly understood. We demonstrate that the uptake of amorphous silica ENPs by macrophage cells, and the secretion of proinflammatory cytokines, is strongly inhibited by silencing expression of scavenger receptor A (SR-A). Conversely, ENP uptake is augmented by introducing SR-A expression into human cells that are normally non-phagocytic. Confocal microscopy analyses show that the majority of single or small clusters of silica ENPs co-localize with SR-A and are internalized through a pathway characteristic of clathrin-dependent endocytosis. In contrast, larger silica ENP agglomerates (>500 nm) are poorly co-localized with the receptor, suggesting that the physical agglomeration state of an ENP influences its cellular trafficking. As SR-A is expressed in macrophages throughout the reticulo-endothelial system, this pathway is likely an important determinant of the biological response to ENPs.

Multiplexed cancer biomarker detection using quartz-based photonic crystal surfaces.

A photonic crystal (PC) surface is demonstrated as a high-sensitivity platform for detection of a panel of 21 cancer biomarker antigens using a sandwich enzyme-linked immunosorbent assay (ELISA) microarray format. A quartz-based PC structure fabricated by nanoimprint lithography, selected for its low autofluorescence, supports two independent optical resonances that simultaneously enable enhancement of fluorescence detection of biomarkers and label-free quantification of the density of antibody capture spots. A detection instrument is demonstrated that supports fluorescence and label-free imaging modalities, with the ability to optimize the fluorescence enhancement factor on a pixel-by-pixel basis throughout the microarray using an angle-scanning approach for the excitation laser that automatically compensates for variability in surface chemistry density and capture spot density. Measurements show that the angle-scanning illumination approach reduces the coefficient of variation of replicate assays by 20-99% compared to ordinary fluorescence microscopy, thus supporting reduction in limits of detectable biomarker concentration. Using the PC resonance, biomarkers in mixed samples were detectable at the lowest concentrations tested (2.1-41 pg/mL), resulting in a three-log range of quantitative detection.

Smoking, COPD, and 3-nitrotyrosine levels of plasma proteins.

Background: Nitric oxide is a physiological regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of smoking. Even so, it is unclear if this effect results from decreased nitric oxide production or increased oxidative degradation of nitric oxide to reactive nitrating species. These two processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. Objective: In this study, we evaluated associations of cigarette smoking and chronic obstructive pulmonary disease (COPD) with nitrotyrosine modifications of specific plasma proteins to gain insight into the processes regulating nitrotyrosine formation. Methods: A custom antibody microarray platform was developed to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. In a cross-sectional study, plasma samples from 458 individuals were analyzed. Results: Average nitrotyrosine levels in plasma proteins were consistently lower in smokers and former smokers than in never smokers but increased in smokers with COPD compared with smokers who had normal lung-function tests. Conclusions: Smoking is associated with a broad decrease in 3-nitrotyrosine levels of plasma proteins, consistent with an inhibitory effect of cigarette smoke on endothelial nitric oxide production. In contrast, we observed higher nitrotyrosine levels in smokers with COPD than in smokers without COPD. This finding is consistent with increased nitration associated with inflammatory processes. This study provides insight into a mechanism through which smoking could induce endothelial dysfunction and increase the risk of cardiovascular disease.

Annexin A2 Modulates Radiation-Sensitive Transcriptional Programming and Cell Fate

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10–200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

Plasma Biomarker Profiles Differ Depending on Breast Cancer Subtype but RANTES Is Consistently Increased

Background: Current biomarkers for breast cancer have little potential for detection. We determined whether breast cancer subtypes influence circulating protein biomarkers. Methods: A sandwich ELISA microarray platform was used to evaluate 23 candidate biomarkers in plasma samples that were obtained from subjects with either benign breast disease or invasive breast cancer. All plasma samples were collected at the time of biopsy, after a referral due to a suspicious screen (e.g., mammography). Cancer samples were evaluated on the basis of breast cancer subtypes, as defined by the HER2 and estrogen receptor statuses. Results: Ten proteins were statistically altered in at least one breast cancer subtype, including four epidermal growth factor receptor ligands, two matrix metalloproteases, two cytokines, and two angiogenic factors. Only one cytokine, RANTES, was significantly increased (P < 0.01 for each analysis) in all four subtypes, with areas under the curve (AUC) for receiver operating characteristic values that ranged from 0.76 to 0.82, depending on cancer subtype. The best AUC values were observed for analyses that combined data from multiple biomarkers, with values ranging from 0.70 to 0.99, depending on the cancer subtype. Although the results for RANTES are consistent with previous publications, the multi-assay results need to be validated in independent sample sets. Conclusions: Different breast cancer subtypes produce distinct biomarker profiles, and circulating protein biomarkers have potential to differentiate between true- and false-positive screens for breast cancer. Impact: Subtype-specific biomarker panels may be useful for detecting breast cancer or as an adjunct assay to improve the accuracy of current screening methods. Cancer Epidemiol Biomarkers Prev; 20(7); 1543–51. ©2011 AACR.

ProMAT calibrator: A tool for reducing experimental bias in antibody microarrays.

Our research group has been developing enzyme-linked immunosorbent assays (ELISA) microarray technology for the rapid and quantitative evaluation of biomarker panels. Studies using antibody microarrays are susceptible to systematic bias from the various steps in the experimental process, and these biases can mask biologically significant differences. For this reason, we have developed a calibration system that can identify and reduce systematic bias due to processing factors. Specifically, we developed a sandwich ELISA for green fluorescent protein (GFP) that is included on each chip. The GFP antigen is spiked into each biological sample or standard mixture and the resulting signal is used for calibration between chips. We developed ProMAT Calibrator, an open-source bioinformatics tool, for the rapid visualization and interpretation of the calibrator data and, if desired, data normalization. We demonstrate that data normalization using this system markedly reduces bias from processing factors. Equally useful, this calibrator system can help reveal the source of the bias, thereby facilitating the elimination of the underlying problem. ProMAT Calibrator can be downloaded at http://www.pnl.gov/statistics/ProMAT .

Line-scanning detection instrument for photonic crystal enhanced fluorescence

A laser line-scanning instrument was developed to optimize the near-field enhancement capability of a one-dimensional photonic crystal (PC) for excitation of surface-bound fluorophores. The excitation laser beam is shaped into an 8 μm × 1 mm line that is focused along the direction of the PC grating, while remaining collimated perpendicular to the grating. Such a beam configuration offers high excitation power density while simultaneously providing high resonant coupling efficiency from the laser to the PC surface. Using a panel of 21 immunofluorescence assays on the PC surface in a microarray format, the approach achieves an enhancement factor as high as 90-fold between on-resonance and off-resonance illumination. The instrument provides a capability for sensitive and inexpensive analysis of cancer biomarkers in clinical applications.

Reducing heterophilic antibody interference in immunoassays using single-chain antibodies

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays can simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as with traditional ELISA diagnostics, endogenous antibodies in patient sera can cause interference. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit (i.e., a single-chain antibody fragment [scFv]) is an effective strategy for reducing assay interference. Our finding illustrates a source of error introduced by the reliance on immunoglobulin-based capture reagents in sandwich immunoassays with human serum samples. We demonstrate that scFvs can be used in such assays to improve reliability by reducing heterophilic antibody interference, thereby improving biomarker analysis and validation.

Application of photonic crystal enhanced fluorescence to antibody microarrays

A photonic crystal surface is utilized for detection of cancer biomarkers in a fluorescent-tagged protein microarray assay. The results indicates that the detection limit of assays are reduced by up to 90% through resonant illumination.