Real-time CRP detection from whole blood using micropost-embedded microfluidic chip incorporated with label-free biosensor.

We demonstrate the detection of C-creative protein (CRP) from whole blood samples without sample pretreatment by using a lab-on-a-chip system consisting of a microfluidic chip and a label-free biosensor. The microfluidic chip includes an array of microposts for filtering blood cells and allows only plasma to flow through and reach the guided-mode resonance (GMR) biosensor for real-time monitoring. The developed GMR sensor can achieve a bulk sensitivity of 186 nm RIU-1, which supports a limit of detection of 3.2 ng mL-1 for recombinant CRP spiked in human serum. The results are comparable with those obtained using enzyme-linked immunosorbent assay. In addition, we demonstrate the efficacy of filtration of blood cells using microposts and simultaneous measurement of CRP concentration using a GMR sensor by using whole blood and plasma samples.

Plant Cell Wall-Penetrable, Redox-Responsive Silica Nanoprobe for the Imaging of Starvation-Induced Vesicle Trafficking.

Autophagy is a self-protection process against reactive oxygen species (ROS). The intracellular level of ROS increased when cells were cultured under nutrient starvation. Antioxidants such as glutathione and ascorbic acid play an important role in ROS removal. However, the cellular redox state in the autophagic pathway is still unclear. Herein, we developed a new redox-sensitive probe with a disulfide-linked silica scaffold to enable the sensing of the reduction environment in cell organelles. This redox-responsive silica nanoprobe (ReSiN) could penetrate the plant cell wall and release fluorescent molecules in response to redox states. By applying the ReSiN to tobacco BY-2 cells and tracing the distribution of fluorescence, we found a higher reducing potential in the central vacuole than in the autolysosomes. Upon cysteine protease inhibitor (E64-c) treatment in sucrose-free medium, the disulfide-silica structures of the ReSiNs were broken down in the vacuoles but were not degraded and were accumulated in the autolysosomes. These results reveal the feasibility of our nanoprobe for monitoring the endocytic and macroautophagic pathways. These pathways merge upstream of the central vacuole, which is the final destination of both pathways. In addition, different redox potentials were observed in the autophagic pathway. Finally, the expression of the autophagy-related protein (Atg8) fused with green fluorescence protein confirmed that the ReSiN treatment itself did not induce the autophagic pathway under normal physiological conditions, indicating the versatility of this nanoprobe in studying stimuli-triggered autophagy-related trafficking.

Compact spectrometer system based on a gradient grating period guided-mode resonance filter.

We demonstrate a compact spectrometer system by using a gradient grating period guided-mode resonance filter-mounted on a linear photodetector array-that exhibits spatially dependent resonance characteristics; a specific incident wavelength is reflected such that the underlying array pixels measure minimum intensity. A precalibrated transmission efficiency matrix is used to determine each pixel's transmission efficiency for specific wavelengths. Unknown spectral information can be calculated from the measured intensity. Grating periods of 250-388 nm in 2-nm increments are used in each 100-cycle period. Device length is 2.23 mm. Spectral range of 506-700 nm is measurable with 1-nm resolution.

Integration of a guided-mode resonance filter with microposts for in-cell protein detection.

We present an integrated microfluidic system consisting of a label-free biosensor of a guided-mode resonance filter (GMRF) and a microfluidic channel with a micropost filter. The GMRF was fabricated through replica molding using an ultraviolet-curable polymer and a plastic substrate. An array of microposts (a diameter and height of 26.5 and 56 µm, respectively, and a spacing between 7.5 and 9.5 µm), fabricated on a silicon substrate through photolithography, was used as the filter. A double-sided tape was used to laminate the GMRF and a microfluidic chip such that the integrated device provides two functions: filtration of the cell debris and quantification of the in-cell protein concentration. By measuring the changes in the resonant wavelength from the GMRF, the detection of ß-actin in an unprocessed lysed cell sample was demonstrated; the cell debris was separated using the micropost filter to prevent false measurement.

Carbon-Based Materials for Photo-Triggered Theranostic Applications.

Carbon-based nanomaterials serve as a type of smart material for photo-triggered disease theranostics. The inherent physicochemical properties of these nanomaterials facilitate their use for less invasive treatments. This review summarizes the properties and applications of materials including fullerene, nanotubes, nanohorns, nanodots and nanographenes for photodynamic nanomedicine in cancer and antimicrobial therapies. Carbon nanomaterials themselves do not usually act as photodynamic therapy (PDT) agents owing to the high hydrophobicity, however, when the surface is passivated or functionalized, these materials become great vehicles for PDT. Moreover, conjugation of carbonaceous nanomaterials with the photosensitizer (PS) and relevant targeting ligands enhances properties such as selectivity, stability, and high quantum yield, making them readily available for versatile biomedical applications.

Effect of high pressure processing on the survival of Shiga toxin-producing Escherichia coli (Big Six vs. O157:H7) in ground beef.

High pressure processing (HPP) is a safe and effective technology for improving food safety. Non-O157:H7 Shiga Toxin-producing Escherichia coli (STEC) have been increasingly implicated in foodborne illness outbreaks and recalls, and the USDA Food Safety Inspection Service (FSIS) has designated them as adulterants in meat (e.g. ground beef). In this study we compared the inactivation of multi-isolate cocktails of E. coli O157:H7 versus the non-O157:H7 STEC "Big Six" (i.e. O26, O45, O103, O111, O121, and O145) in ground beef (83% lean) using HPP at refrigeration temperature (4-7 °C). A >5-log CFU/g inactivation of both the Big Six and O157:H7 cocktails were observed at 450 MPa for 15 min. In general, the Big Six cocktail was found more sensitive to pressure stress (p < 0.05). In contrast, HPP treatment at 250 MPa (30 min) inactivated only 2.3 log of the Big Six versus 1.0 log of O157:H7. HPP treatment at 350 MPa (30 min) inactivated 4.7 log of the Big Six vs. 3.2 log of O157:H7. Multiple-cycle HPP cycles (250 or 350 MPa, three 5 min treatments) did not result in a 5 log reduction of the non-O157:H7 or O157:H7 STEC. Our results indicate that HPP inactivation parameters which are effective for O157:H7 STEC can be used for the non-O157:H7 Big Six isolates in ground beef.

Effect of high pressure treatment on the survival of Shiga toxin-producing Escherichia coli in strawberry puree.

Most fresh produce, such as strawberries, receives minimal processing and is often eaten raw. Contamination of produce with pathogenic bacteria may occur during growth, harvest, processing, transportation, and storage (abuse temperature) and presents a serious public health risk. Strawberries have been implicated in an outbreak of Escherichia coli O157:H7 infection that sickened 15 people, including one death. Strawberries may also be contaminated by other serogroups of non-O157 Shiga toxin-producing E. coli (STEC), including O26, O45, O103, O111, O121 and O145, which have become known as the "Big Six" or "Top Six" non-O157 STECs. The objective of this research was to explore the potential application of high pressure processing (HPP) treatment to reduce or eliminate STECs in fresh strawberry puree (FSP). FSP, inoculated with a six-strain cocktail of the "Big Six" non-O157 STEC strains or a five-strain cocktail of E. coli O157:H7 in vacuum-sealed packages, were pressure-treated at 150, 250, 350, 450, 550, and 650 MPa (1 MPa = 10(6) N/m(2)) for 5, 15, and 30 min. HPP treatment, at 350 MPa for ≥5 min, significantly reduced STECs in FSP by about 6-log CFU/g from the initial cell population of ca. 8-log CFU/g. Cell rupture, observed by scanning electron microscopy (SEM), demonstrated that the HPP treatments can be potentially used to control both non-O157 and O157:H7 STECs in heat sensitive products.

Non-metallic nanomaterials in cancer theranostics: a review of silica- and carbon-based drug delivery systems.

The rapid development in nanomaterials has brought great opportunities to cancer theranostics, which aims to combine diagnostics and therapy for cancer treatment and thereby improve the healthcare of patients. In this review we focus on the recent progress of several cancer theranostic strategies using mesoporous silica nanoparticles and carbon-based nanomaterials. Silicon and carbon are both group IV elements; they have been the most abundant and significant non-metallic substances in human life. Their intrinsic physical/chemical properties are of critical importance in the fabrication of multifunctional drug delivery systems. Responsive nanocarriers constructed using these nanomaterials have been promising in cancer-specific theranostics during the past decade. In all cases, either a controlled texture or the chemical functionalization is coupled with adaptive properties, such as pH-, light-, redox- and magnetic field- triggered responses. Several studies in cells and mice models have implied their underlying therapeutic efficacy; however, detailed and long-term in vivo clinical evaluations are certainly required to make these bench-made materials compatible in real bedside circumstances.

Application of Nanosecond Pulsed Electric Field and Autofluorescence Lifetime Microscopy of FAD in Lung Cells.

Exposure of nanosecond pulsed electric fields (nsPEFs) to live cells is an increasing research interest in biology and medicine. Despite extensive studies, a question still remains as to how effects of application of nsPEF on intracellular functions are different between cancerous cells and normal cells and how the difference can be detected. Herein, we have presented an approach of autofluorescence lifetime (AFL) microscopy of flavin adenine dinucleotide (FAD) to detect effects of application of nsPEF having 50 ns of a pulse width, nsPEF(50), on intracellular function in lung cancerous cells, A549 and H661, which show nsPEF(50)-induced apoptosis, and normal cells, MRC-5, in which the field effect is less or not induced. Then, the application of nsPEF(50) is shown to increase the lifetime of FAD autofluorescence in lung cancerous cells, whereas the electric field effects on the autofluorescence of FAD was not significant in normal healthy cells, which indicates that the lifetime measurements of FAD autofluorescence are applicable to detect the field-induced change in intracellular functions. Lifetime and intensity microscopic images of FAD autofluorescence in these lung cells were also acquired after exposure to the apoptosis-inducer staurosporine (STS). Then, it was found that the AFL of FAD became longer after exposure not only in the cancerous cells but also in the normal cells. These results indicate that nsPEF(50) applied to lung cells induced apoptotic cell death only in lung cancerous cells (H661 and A549) but not in lung normal cells (MRC-5), whereas STS induced apoptotic cell death both in lung cancerous cells and in lung normal cells. The lifetime microscopy of FAD autofluorescence is suggested to be very useful as a sensitive detection method of nsPEF-induced apoptotic cell death.

Multiplexed immunoassays for the analysis of breast cancer biopsies.

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated.

Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells.

Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time-resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC-5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue- and red-shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra-cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.

Artificial peptide-controlled protein release of Zn2+-triggered, self-assembled histidine-tagged protein microparticle.

Protein microparticles have received attention as drug delivery systems because of their high protein stability and prolonged release in vivo. However, most current preparation processes introduce chemical crosslinkers, which often lead to protein inactivation and limit drug efficacy in delivery systems. In this study, we employed the well-known hexahistidine (His)-tag recombinant protein technology and a metal-triggerable collagen-mimetic peptide to enhance the binding strength between the protein and metal ion and fine-tuned the protein drug release. His-tagged proteins self-assembled to form microparticles (∼2 µm) upon zinc ion treatment and sustained protein drug release was achieved in physiological saline. The results also indicated that by adjusting the peptide concentration and N- and C-terminal hexahistidine-tags, protein release could be controlled. Moreover, no protein denaturation was observed. We developed a universal strategy for facile protein microparticle fabrication under mild conditions and we demonstrated its potential as a therapeutics formulation with tunable protein release.

Diatom-assisted biomicroreactor targeting the complete removal of perfluorinated compounds.

Persistent perfluorinated compounds (PFCs) have been recognized as a global environmental issue. Developing methods without leading to additional burden in nature will be essential for PFCs removal. Herein, we functionalized iron nanoparticles on living diatom (Dt) to efficiently enable the Fenton reaction and reactive oxygen species (ROS) production. Iron nanoparticles at the surface of living diatom act as promising catalytic agents to trigger OH radical generation from H2O2. Dt plays dual roles: i) as solid support for effective adsorption, and ii) it supplies oxygen and inherently produces ROS under stress conditions, which improves removal efficiency of PFCs. We also demonstrated its reusability by simple magnetic separation and 85% of decomposition efficiency could still be achieved. This newly developed diatom-assisted bioremediation strategy enables green and efficient PFC decomposition and shall be readily applicable to other persistent pollutants.

Longitudinal and quantitative assessment platform for concurrent analysis of anti-tumor efficacy and cardiotoxicity of nano-formulated medication in vivo.

Increasing nanomedicinal approaches have been developed to effectively inhibit tumor growth; however, critical questions such as whether a nanomedicinal approach can mitigate latent side effects are barely addressed. To this end, we established a zebrafish xenograft tumor model, combining pseudodynamic three-dimensional cardiac imaging and image analysis to enable simultaneous and quantitative determination of the change of tumor volume and cardiac function of zebrafish upon specific nanoformulation treatment. Doxorubicin (DOX), a well-known chemotherapeutic agent with cardiotoxicity, and a recently developed DOX-loaded nanocomposite were employed as two model drugs to demonstrate the effectiveness to utilize the proposed evaluation platform for rapid validation. The nanoformulation significantly mitigated DOX-associated cardiotoxicity, while retaining the efficacy of DOX in inhibiting tumor growth compared to administration of carrier-free DOX at the same dose. We anticipate that this platform possesses the potential as an efficient assessment system for nanoformulated cancer therapeutics with suspected toxicity and side effects to vital organs such as the heart.

Palladium-catalyzed α-arylation of indolin-3-ones.

A method for the catalytic α-arylation of indolin-3-ones was developed. The catalytic system comprising Pd(dba)2 and PAd3 was found to be optimal for the transformation. The protocol features broad functional group compatibility in that a range of arylated indoxyl derivatives bearing a fully substituted carbon center was synthesized with high efficiency. A preliminary bioassay study revealed that the selected indole-substituted indolin-3-ones exhibit favorable cytotoxic activities against HCT-116 cancer cell line.

Multiplex microsphere-based flow cytometric platforms for protein analysis and their application in clinical proteomics - from assays to results.

Advances in high-throughput screening, together with rapid progress in genomic and proteomic sciences, have considerably stimulated the development of a variety of biomarker discovery tools and have contributed to the improvement of clinical diagnosis. Major challenges include the obtaining of high-quality data sets based on assays that are rapid, reliable, and inexpensive, but still highly sensitive and easy to perform. The microsphere-based flow cytometric technology is a platform that is currently entering the diagnostic market. This article reviews the development and the applications of microsphere-based flow cytometric platforms in basic and applied proteomic research, and discusses bead-based assays used in clinical applications.

Pathogen specific cytokine release reveals an effect of TLR2 Arg753Gln during Candida sepsis in humans.

Toll-like receptors (TLRs) are crucial pattern-recognition receptors (PRRs) for activation of innate and adapted immunity. TLR2 heterodimerizes with TLR1 or TLR6 to recognize multiple pathogen-associated molecular patterns (PAMPs) of fungi, Gram-positive pathogens, and mycobacteria. Receptor activation culminates in monocyte, T-helper (Th)1, and Th2 cytokine release. Single nucleotide polymorphisms (SNPs) Arg753Gln and Arg677Trp affect TLR2 responsiveness and may contribute to the course of sepsis, which is associated with substantial morbidity and mortality during intensive care treatment. We genotyped 325 critically ill patients with septic shock, and performed a detailed clinical follow-up with 47 of these patients. Here, we investigated whether distinct sepsis episodes result in defined plasma cytokine patterns, and whether cytokine profiles may be linked to the TLR2 polymorphisms. Blood sampling was done daily and microbiological testing was performed on a routine basis. DNA was extracted from whole blood and TLR2 SNPs were typed by pyrosequencing. Cytokines were measured by multiplexed array technologies and the leukocyte phenotype was determined by flow cytometry. Among the 325 ICU patients, 17 individuals (5.2%) were heterozygous for Arg753Gln. The SNP Arg677Trp was not found in any patient. Episodes of Gram-negative, Gram-positive, and Candida sepsis were recorded. During Gram-positive sepsis, the cytokine pattern did not differ between Arg753Gln heterozygous patients and wild type patients. By contrast, during Candida sepsis, the Arg753Gln heterozygous patients showed biomarker patterns that differed from wild type patients with elevated TNF-alpha plasma concentrations, but reduced IFN-gamma and IL-8 levels. In conclusion, TLR2 SNP Arg753Gln results in altered cytokine release in response to Candida but not to Gram-positive sepsis.

Miniaturized parallelized sandwich immunoassays.

This chapter describes the development and use of bead-based miniaturized multiplexed sandwich immunoassays for focused protein profiling. Bead-based protein arrays or suspension microarrays allow simultaneous analysis of a variety of parameters within a single experiment. In suspension microarrays capture antibodies are coupled onto color-coded microspheres. The applications of suspension microarrays are described, which allow to analyze proteins present in different types of body fluids, such as serum or plasma, cerebrospinal, pleural and synovial fluids, as well as cell culture supernatants. The chapter is divided into the generation of suspension microarrays, sample preparation, processing of suspension microarrays, validation of analytical performance, and finally pattern generation using bioinformatics tools.

Suspension microarrays for the identification of the response patterns in hyperinflammatory diseases.

Miniaturized and parallelized sandwich immunoassays allow the simultaneous analysis of a variety of parameters in a single experiment. Bead-based protein array systems or suspension microarrays are well-established multiplex sandwich immunoassay formats. To study inflammatory diseases, protein arrays can be used to analyze changes in plasma protein levels, such as cytokines, chemokines, soluble receptors, and matrix metalloproteinases. Using the bead-based Luminex system, multiplexed sandwich immunoassays have been developed to analyze the plasma concentrations of soluble receptors: sTNF-RI, sTNF-RII, sIL-2R, sgp130, sFas, sRAGE, sE-selectin, sICAM-1, sVCAM-1, sMIF-1 and sFasL. This newly established 11-plex soluble receptors assay demonstrated acceptable intra-assay and inter-assay precision, appropriate accuracy, and no crossreactivity between analytes. Using this assay, 100 plasma samples derived from 36 critically ill intensive care unit (ICU) patients with trauma or sepsis were analyzed for their soluble receptor plasma concentrations. Results obtained allowed grouping of patients' samples into a trauma and a sepsis group. Four candidate molecules: sFas, sICAM-1, sTNF-RI, and sTNF-RII had higher concentrations in patients with sepsis than in those with trauma, contributing the highest discriminatory values to define the nature of the inflammatory disease originating from pathogen-involved (sepsis) or pathogen-independent inflammation.

A protein detection technique by using surface plasmon resonance (SPR) with rolling circle amplification (RCA) and nanogold-modified tags.

Surface plasmon resonance (SPR) can detect molecules bound to a surface by subtle changes in the SPR angle. By immobilizing probes onto the surface and passing analyte solution through the surface, changes in SPR angle indicate the binding between analyte and probes. Detection of analyte from solution can be achieved easily. By using rolling circle amplification (RCA) and nanogold-modified tags, the signals of analyte binding are greatly amplified, and the sensitivity of this technique is significantly improved. Furthermore, this technique has potentials for ultra-sensitive detection and microarray analysis. In this paper, this detection technique is introduced and shown to have great amplification capability. Using 5 nm nanogold with 30 min of RCA development time, this proposed protein detection technique shows over 60 times amplification of the original signal.