A cell array biosensor for environmental toxicity analysis

In this study, a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity has been implemented to develop two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 microl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.     

用于药物筛选的微流控细胞阵列芯片

细胞区域分布培养以及如何有效地对微流体进行操控是微流控阵列芯片在细胞药物研究中的关键技术。本研究介绍了一种利用SU-8负性光刻胶模具和PDMS制作双层结构的微流控细胞阵列芯片的方法,该芯片通过C型的坝结构将进样细胞拦截在芯片的细胞培养的固定区域,键合双层PDMS构成阀控制层,阀网络的开关作用成功实现了芯片通道内微流体的操控,同时芯片设计了药物浓度梯度网络,产生6个不同浓度的药物刺激细胞。通过对芯片3种共培养细胞活性的检测和药物伊立替康(CTP-11)对肝癌细胞的浓度梯度刺激等实验结果验证该芯片在细胞研究和药物筛选等方面的可行性。     

Micropillar array chip for integrated white blood cell isolation and PCR

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 microm in cross-section, etched to a depth of 80 microm with adjacent pillars being separated by 3.5 microm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip.     

Amperometric detection of the bacterial metabolic regulation with a microbial array chip

A microbial array chip with collagen gel spots entrapping living bacterial cells has been applied to investigate the metabolic regulation in Paracoccus denitrificans. Scanning electrochemical microscopy (SECM) was used to monitor the ferrocyanide production that reflects the electron flow in the respiratory chain located within the internal membrane of P. denitrificans. The ferrocyanide production from P. denitrificans largely depends on the types of the carbon source (glucose or lactate), suggesting that the electron flow rate in the respiratory chain depends on the activity of the metabolic pathway located up-stream of the respiratory chain. More importantly, it was found that the enzymes affecting glucose catabolic reactions were significantly up-regulated in cultures with a nutrient agar medium containing D-(+)-glucose as a sole carbon source. Enzyme assays using crude extracts of P. denitrificans were carried out to identify the enzymes expressed at a higher level in cultures supplemented with D-(+)-glucose. It was confirmed that the pyruvate kinase and enzymes of the overall Entner-Doudoroff pathway were highly induced in cultures containing D-(+)-glucose.     

Optical imaging fiber-based live bacterial cell array biosensor

A live cell array biosensor was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high-density array of microwells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specific analyte. A genetically modified Escherichia coli strain, containing the lacZ reporter gene fused to the heavy metal-responsive gene promoter zntA, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell lacZ expression was measured when the array was exposed to mercury and a response to 100nM Hg(2+) could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.     

An insect model for assessing oxidative stress related to arsenic toxicity

The potential usefulness of an insect model to evaluate oxidative stress induced by environmental pollutants was examined with trivalent arsenic (As³⁺, NaAsO₂) and pentavalent arsenic (As⁵⁺, Na₂HAsO₄) in adult female house flies, Musca domestica, and fourth-instar cabbage loopers, Trichoplusia ni. M. domestica was highly susceptible to both forms of arsenic following 48 h exposure in the drinking water with LC₅₀s of 0.008 and 0.011% w/v for As³⁺ and As⁵⁺, respectively. T. ni larvae were susceptible to dietary As³⁺ with an LC₅₀ of 0.032% w/w but seem to tolerate As⁵⁺ well with an LC₅₀ of 0.794% concentration after 48 h exposure. The minimally acute LC₅ dose of both As³⁺ and As⁵⁺ varied considerably but averaged 0.005% for both insects. The potential of both valencies of arsenic for inducing oxidative stress in the insects exposed ad libitum to approximately LC₅ levels was assessed. The parameters examined were the alterations of the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), the peroxidase activity of glutathione transferase (GSTPX), and glutathione reductase (GR), and increases in lipid peroxidation and protein oxidation. SOD (1.3-fold), GST (1.6-fold), and GR (1.5-fold) were induced by As³⁺ in M. domestica but CAT and GSTPX were not affected. As⁵⁺ had no effect on M. domestica. In T. ni, the antioxidant enzyme activities were not affected by As³⁺ except for SOD which was suppressed by 29.4% and GST which was induced by 1.4-fold. As⁵⁺ had no effect except the suppression of SOD by 41.2%. Lipid peroxidation and protein oxidation, which represent stronger indices of oxidative stress, were elevated in both insects by up to 2.9-fold. However, based on the antioxidant enzyme response to the arsenic anions, the mode of action of arsenic induced oxidative stress may differ between the two insects. Until this aspect is further clarified, evidence at this time favors the prospect of As³⁺ as a pro-oxidant, especially for M. domestica. © 1995 Wiley-Liss, Inc.     

An inexpensive infrared growth sensor array for detection of bacterial antibiotic susceptibility

Abstract An inexpensive infrared sensor was constructed and used for the rapid testing of bacterial antibiotic susceptibility by detection of changes in absorbance at 950 nm. By comparing cultures of clinical isolates together with control strains ( Escherichia coli NCTC 10418, Staphylococcus aureus NCTC 6571 or Pseudomonas aeruginosa NCTC 10662) after addition of an antibiotic, results on susceptibility were obtained within 3–5 h from the original plate culture. Representative strains of E. coli, P. aeruginosa , and S. aureus were tested successfully against ampicillin, penicillin, gentamicin or ciprofloxacin.     

A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.     

Facile synthesis of metal-chelating peptides on chip for protein array

A unique peptide sequence of HGGHHG screening from a combinatorial synthetic peptide library showed a good chelating ability to bind a transition metal on a chip better than hexa-His peptide. It was directly conjugated with a His-Tagged proteins onto a chip in a mild aqueous solution and can be used this chip as a high throughput technique for protein array in order to detect and purify the His-Tagged proteins.     

A novel cell array technique for high-throughput,cell-based analysis

Summary Microarray technology has burgeoned over the past few years from nucleic acid-based arrays to tissue microarrays (TMAs). This study aimed to develop a technique to incorporate cell lines into an array and to demonstrate the usefulness of this technique by performing immunohistochemistry for β-catenin. Cell suspensions were prepared from 23 tumor cell lines. These were fixed in formalin, suspended in agar, and embedded in paraffin to produce a cell block. A “tissue microarrayer” was used to remove triplicate, 0.6 mm-cores from each cell block and to transfer these into a recipient paraffin block at precise coordinates. Immunohistochemistry was used to identify cell lines positive for β-catenin. Cultured cells were successfully incorporated into the microarray, with preservation of cell architecture and even distribution of cells within each core. A total of 18 of 69 cores (26%) were lost in processing. A total of 16 of 23 cell lines were identified as positive for membrane and cytoplasmic β-catenin, and 6 of 23 were negative. Only one cell line was unscorable because of complete core loss. We have developed a “cell microarray” technique for analyzing antigen expression by immunohistochemistry in multiple cell lines in a single expriment. This novel application of microarrays permits high-throughput, cost-efficient analysis, with the potential to rapidly identify markers with potential diagnostic and therapeutic implications in human disease.     

The role of reactive oxygen species and oxidative stress in carbon monoxide toxicity: An in-depth analysis

Abstract

The underlying mechanism of the central nervous system (CNS) injury after acute carbon monoxide (CO) poisoning is interlaced with multiple factors including apoptosis, abnormal inflammatory responses, hypoxia, and ischemia/reperfusion-like problems. One of the current hypotheses with regard to the molecular mechanism of CO poisoning is the oxidative injury induced by reactive oxygen species, free radicals, and neuronal nitric oxide. Up to now, the relevant mechanism of this injury remains poorly understood. The weakening of antioxidant systems and the increase of lipid peroxidation in the CNS have been implicated, however. Accordingly, in this review, we will highlight the relationship between oxidative stress and CO poisoning from the perspective of forensic toxicology and molecular toxicology.     

An acoustic method for the analysis of bacterial cells

The application of a biological electroacoustic sensor based on a lateral electric-field-excited piezoelectric resonator for the study of bacterial cells that interact with specific bacteriophages, mini-antibodies, and polyclonal antibodies was successfully demonstrated. The determined lower limit of microbialcell detection was approximately of 10³ to 10⁴ cells/mL for the duration of the assay of 10 min. The possibility of bacterial-cell detection via interaction with specific agents in the presence of extraneous microbiota was shown. The method allowed us to determine the spectrum of lytic activity of bacteriophages and the sensitivity of microbial cells to bacteriophages. The results of the study showed that application of a sensor piezoelectric lateral-field resonator is a promising technique for the detection and identification of microbial cells and determination of their phage resistance in microbiology, medicine, and veterinary medicine. Furthermore, the results of the experiments made it possible to understand the mechanisms of the processes that occur in a suspension of bacterial cells in the presence of various biological agents. The method also may provide useful information regarding biophysical mechanisms of interactions that occur in microbial populations.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

An associative analysis of gene expression array data

MOTIVATION: We face the absence of optimized standards to guide normalization, comparative analysis, and interpretation of data sets. One aspect of this is that current methods of statistical analysis do not adequately utilize the information inherent in the large data sets generated in a microarray experiment and require a tradeoff between detection sensitivity and specificity. RESULTS: We present a multistep procedure for analysis of mRNA expression data obtained from cDNA array methods. To identify and classify differentially expressed genes, results from standard paired t-test of normalized data are compared with those from a novel method, denoted an associative analysis. This method associates experimental gene expressions presented as residuals in regression analysis against control averaged expressions to a common standard-the family of similarly computed residuals for low variability genes derived from control experiments. By associating changes in expression of a given gene to a large family of equally expressed genes of the control group, this method utilizes the large data sets inherent in microarray experiments to increase both specificity and sensitivity. The overall procedure is illustrated by tabulation of genes whose expression differs significantly between Snell dwarf mice (dw/dw) and their phenotypically normal littermates (dw/+, +/+). Of the 2,352 genes examined only 450-500 were expressed above the background levels observed in nonexpressed genes and of these 120 were established as differentially expressed in dwarf mice at a significance level that excludes appearance of false positive determinations.     

An array of microfabricated pillars to study cell migration

Mechanical forces play an important role in various cellular functions, such as tumor metastasis, embryonic development or tissue formation. Cell migration involves dynamics of adhesive processes and cytoskeleton remodelling, leading to traction forces between the cells and their surrounding extracellular medium. To study these mechanical forces, a number of methods have been developed to calculate tractions at the interface between the cell and the substrate by tracking the displacements of beads or microfabricated markers embedded in continuous deformable gels. These studies have provided the first reliable estimation of the traction forces under individual migrating cells. We have developed a new force sensor made of a dense array of soft micron-size pillars microfabricated using microelectronics techniques. This approach uses elastomeric substrates that are micropatterned by using a combination of hard and soft lithography. Traction forces are determined in real time by analyzing the deflections of each micropillar with an optical microscope. Indeed, the deflection is directly proportional to the force in the linear regime of small deformations. Epithelial cells are cultured on our substrates coated with extracellular matrix protein. First, we have characterized temporal and spatial distributions of traction forces of a cellular assembly. Forces are found to depend on their relative position in the monolayer : the strongest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. Consequently, these forces are quantified and correlated with the adhesion/scattering processes of the cells.