Colorimetric Detection System Research Articles

Hemin−Graphene Hybrid Nanosheets with Intrinsic Peroxidase-like Activity for Label-free Colorimetric Detection of Single-Nucleotide Polymorphism

This paper demonstrated for the first time a simple wet-chemical strategy for synthesizing hemin-graphene hybrid nanosheets (H-GNs) through the π-π interactions. Significantly, this new material possesses the advantages of both hemin and graphene and exhibits three interesting properties. First, H-GNs have intrinsic peroxidase-like activity, which can catalyze the reaction of peroxidase substrate, due to the existence of hemin on the graphene surface. Second, their dispersion follow the 2D Schulze-Hardy rule, that is to say, the coagulation of H-GNs in electrolyte solution results from the interplay between van der Waals attraction and electric double-layer repulsion. Third, H-GNs exhibit the ability to differentiate ss- and ds-DNA in optimum electrolyte concentration, owing to the different affinities of ss- and ds-DNA to the H-GNs. On the basis of these unique properties of the as-prepared H-GNs, we have developed a label-free colorimetric detection system for single-nucleotide polymorphisms (SNPs) in disease-associated DNA. To our knowledge, this is the first report concerning on SNPs detection using functionalized graphene nanosheets. Owing to its easy operation and high specificity, it was expected that the proposed procedure might hold great promise in the pathogenic diagnosis and genetic diseases.

Rapid Detection of Enterotoxigenic Escherichia coli Gene Using Bio-Conjugated Gold Nano-Particles

In this study, a simple gold nanoparticle based colorimetric detection system for on site diagnostics of Enterotoxigenic Escherichia coli (ETEC) has been developed. The developed assay could detect LT1 gene as evident from UV-visible spectra, the broadening of peak on hybridization of GNP probes with different copy numbers of the target gene. The present study suggests that hybridization induced changes in optical properties of GNPs can be translated into a rapid and simple colorometric" spot and read test detection method for ETEC.

Ultra-sensitive detection of cysteine by gold nanorod assembly

In this paper, we have developed a simple, rapid, selective, and ultra-sensitive assay to detect cysteine by means of gold nanorods assembly. This methodology is based on the end-to-end assembly of gold nanorods and it is very sensitive due to the useful formation of the gold nanorods chain in the presence of cysteine under acidic aqueous solution. The sharp absorption peak changes observed from the assembly formed by the gold nanorods allow one to monitor very subtle differences induced by cysteine in an acidic medium and therefore, it can provide a quantitative measure of the cysteine concentration. On the other hand, the high extinction coefficients of gold nanorods allow one to monitor the changes at lower concentrations compared to that accomplished by conventional absorbance-based chromophores. Therefore, one of the significant features is the ability to detect cysteine in the presence of other essential α-amino acids at concentrations as low as 10 pM, which to the best of our knowledge is the lowest reported for a colorimetric cysteine detection system. More importantly, this assay is particularly attractive because it does not rely on organic cosolvents, enzymatic reactions, light-sensitive dye molecules, lengthy protocols, and sophisticated instrumentation.

Effects of melatonin on wortmannin-induced tau hyperphosphorylation

To explore the underlying mechanism of tau hyperphosphorylation in an Alzheimeros-affected brain and the possible arresting strategies. MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide), crystal violet assay, phase-contrast, dead end colorimetric apoptosis detection system (TUNEL) and electron microscopy were used to detect cell viability, morphology and apoptosis. Western blot, 32P-labeling and the detection of malondialdehyde level and superoxide dismutase activity were used respectively for the phosphorylation level of tau, the activity of glycogen synthase kinase (GSK-3), and oxidative stress measurement. Exposure of the cells to wortmannin resulted in an obvious lipid peroxidation, reduction of cell viability, cell process retraction, and plasma vacuolation, but with no obvious cell apoptosis. We also found that preincubation of the cells with melatonin or vitamin E attenuated differentially wortmannin-induced oxidative stress as well as GSK-3 overactivation and tau hyperphosphorylation. Wortmannin is an effective tool for reproducing Alzheimer-like tau hyperphosphorylation cell model and melatonin/vitamin E can effectively protect the cells from wortmannin-induced impairments.

Induction of thymine dimers by solar radiation in natural freshwater phytoplankton assemblages in Patagonia, Argentina

Natural phytoplankton assemblages from a freshwater lake in Trelew (Province of Chubut, Patagonia, Argentina) were exposed to natural solar radiation at different depths in a water basin filled with fresh water rich in humic substances. Samples were taken at regular intervals for DNA extraction and subsequent analysis of DNA damage by determining the formation of thymine dimers using an immuno-dot-blot procedure. The use of a colorimetric detection system based on the reaction of alkaline phosphatase with BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitro blue tetrazolium) proved unsatisfactory because of uneven and strong background signals. As a consequence the antibodies were stripped and the blots were reprobed successfully, using a chemoluminescence based detection system. Natural solar radiation caused an increase from 3.2 ± 0.9 thymine dimers (T^T) per mega base pair (Mbp) to 50.9 ± 3.8 T^T Mbp−1 during the light period in natural phytoplankton exposed at the surface. When the UV-B component of the solar radiation was removed by a 320 nm cut-off filter, no thymine dimers were produced. After prolonged exposure at the surface, some but not total recovery occurred overnight. There was no significant DNA damage in the samples exposed at 85 cm depth (kd = 3.62 m−1 at 305 nm). The results indicate that the impinging UV-B radiation at the study site may be deleterious to the survival of phytoplankton exposed at the water surface and thus significantly alter the nutrient cycling and biomass production.

Eugenol Causes Melanoma Growth Suppression through Inhibition of E2F1 Transcriptional Activity

Metastatic malignant melanoma is an extremely aggressive cancer, with no currently viable therapy. 4-Allyl-2-methoxyphenol (eugenol) was tested for its ability to inhibit proliferation of melanoma cells. Eugenol but not its isomer, isoeugenol (2-methoxy-4-propenylphenol), was found to be a potent inhibitor of melanoma cell proliferation. In a B16 xenograft study, eugenol treatment produced a significant tumor growth delay (p = 0.0057), an almost 40% decrease in tumor size, and a 19% increase in the median time to end point. More significantly, 50% of the animals in the control group died from metastatic growth, whereas none in the treatment group showed any signs of invasion or metastasis. Eugenol was well tolerated as determined by measurement of bodyweights. Examination of the mechanism of the antiproliferative action of eugenol in the human malignant melanoma cell line, WM1205Lu, showed that it arrests cells in the S phase of the cell cycle. Flow cytometry coupled with biochemical analyses demonstrated that eugenol induced apoptosis. cDNA array analysis showed that eugenol caused deregulation of the E2F family of transcription factors. Transient transfection assays and electrophoretic mobility shift assays showed that eugenol inhibits the transcriptional activity of E2F1. Overexpression of E2F1 restored about 75% of proliferation ability in cultures. These results indicate that deregulation of E2F1 may be a key factor in eugenol-mediated melanoma growth inhibition both in vitro and in vivo. Since the E2F transcription factors provide growth impetus for the continuous proliferation of melanoma cells, these results suggest that eugenol could be developed as an E2F-targeted agent for melanoma treatment.

Detection of Mycobacterium Avium Subsp. Paratuberculosis in Bovine Fecal Samples: Comparison of Three Polymerase Chain Reaction—Based Diagnostic Tests with a Conventional Culture Method

Three commercially available assays, designed to specifically detect the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal samples by IS900-PCR, were compared with a conventional culture method. Fecal samples from 100 dairy cows were tested. Fifty-four (67.5%) of 80 culture-positive samples were positive for an assay that detects MAP DNA by dot spot hybridization of polymerase chain reaction products (kit A), 48 (60%) were positive by an assay using ethidium bromide staining for agar gel visualization of amplification products (kit B), and 49 (61.3%) were positive by an assay in which amplified products are detected by a colorimetric detection system (kit C). Relative sensitivity of all tests increased in proportion to the presence of MAP in fecal samples. Specificity was 100% based on results from 20 culture-negative samples from an MAP-free herd.

Development of a High-Throughput Plate-Based Chemiluminescent Transcription Factor Assay

Transcription factors are DNA-binding proteins that regulate the expression of specific genes by controlling transcription initiation. Two families of transcription factors, NFkappaB and AP-1, play pivotal roles in controlling important cellular processes ranging from normal cell growth and differentiation to apoptosis and cancer. Identifying changes in the DNA-binding activity of these factors is essential to understanding the regulation of these processes. We have developed a high-throughput DNA-based ELISA capable of monitoring activated levels of NFkappaB (p50 and p65) and AP-1 (c-Jun and c-Fos). This chemiluminescent assay utilizes a 96-well plate format, eliminating the throughput challenges imposed by traditional gel shift assays and exceeding the sensitivity and dynamic range of standard colorimetric detection systems. The sensitivity of this assay enables distinction between subtle as well as dramatic differences in the DNA-binding activity of these factors that result from the treatment of cells with various inhibitors or activating agents.

Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch hydrolysis.

High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of reducing agents, particularly the glucose used in yeast growth media. The present investigation employed colorimetric and chemiluminescent detection systems that enable direct and rapid screening of activities on raw starch substrate. Active clones could be separated into two groups, based on high total activity or high specific activity.

PCR-reverse line blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvement in Lyme disease.

Detection of the Borrelia burgdorferi sensu lato complex in biological samples is currently done by conventional immunological and molecular biological methods. To improve on the accuracy of these methods and to simplify the procedure for testing large numbers of samples, a solid-phase sandwich hybridization system readily applicable to the detection of PCR products has been designed. This colorimetric detection system relies on the use of polybiotinylated detection probes and of specific capture oligonucleotides covalently linked at allocated positions on nylon membrane strips. From a phylogenetic analysis on a great number of ospA gene sequences, we have designed and synthesized a set of PCR primers specific to the five Borrelia burgdorferi sensu lato genospecies present in Europe and a subset of probes (capture and detection probes) specific to these five genospecies (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae). This combined PCR hybridization system was evaluated with a large number of various B. burgdorferi isolates and clinical specimens. These analyses clearly showed that the system could be used as a typing method to distinguish five genospecies belonging to the B. burgdorferi sensu lato complex. In addition, the study showed that B. valaisiana strains might be more heterologous than suspected up to now and clustered into three genomic groups.

Identification of endometriosis-related genes by cDNA microarray

Objective: Endometriosis is the most common cause of female infertility and too often the molecular mechanisms is still unclear. This study aims to identify differential expressed genes in eutopic endometrium (uterine endometrium) versus ectopic endometrium (chocolate cyst) through the use of a membrane-based cDNA microarray. Design: Pilot study. Materials/Methods: Patients who were scheduled for surgery for endometriosis were recruited to participate in this study. Eutopic endometrial tissues from women with endometriosis (n=3) and the ectopic endometrial tissues from chocolate cysts (n=3) were collected during laparoscopy. The tissues were irrigated with lactated Ringer’s solution and divided in half. One half was immediately placed in an RNA protection reagent, RNAlater (Ambion, Austin, TX) and placed on ice for transport to the laboratory. The other half was sent to the pathology laboratory. The human cDNA microarray system (9,600 genes, including known regulatory genes and expressed sequence tag, EST) with colorimetric detection system was used to identify the differentially expressed genes. Results: The results of cDNA expression array showed that 163 genes were up-regulated 3-fold or higher in ectopic endometrium and only 14 genes were up-regulated in eutopic endometrium at the mRNA level. These differentially expressed genes were further grouped into categories by their putative functions, including: cell growth-related factors, cell adhesion molecules, signal transduction molecules, apoptotsis-related factors, hormones/cytokines, cytoskeleton/extracellular matrix proteins, and some expressed sequence tags (ESTs). Six of the genes that belong to the categories of cell apoptosis (Bcl-2, 4.6 folds), cell cycle-related genes (cyclin B1, 3.6 folds), cell migration and invasion (Vascular cell adhesion molecule 1, VCAM-1, 3.6 folds), growth factors/cytokines (BMP-4, 3.0 folds and TGF-beta receptor, 5.8 folds), metabolism (Glutathione S-transferase A2, GSTA2, 7.1 folds), and immune-response (CD36, 5.9 folds) were studied more extensively for their protein expression by immunohistochemistry. The immunostaining results showed that Bcl-2, cyclin B1, VCAM-1, BMP-4, TGF-beta receptor, GSTA2, and CD36 were over-expressed in ectopic endometrium at the protein level, validating the microarray findings. Conclusions: The globally analysis of the gene regulation in endometriosis was reported for the first time in this study. According to the results, we found that several genes related to cell invasion, cell growth, and immuno-response were highly regulated in ectopic endometrium. Overall, this study demonstrates that several genes are differentially expressed in ectopic endometrium. It is helpful to identify gene expression markers of endometriosis that can be used for noninvasive diagnosis of endometriosis, for development of new treatments for endometriosis, or to gain an understanding of the pathophysiology and etiology of the disease. Supported by: This investigation was supported by grants from the National Science Council (NSC 90-2314-B-038-049).

Apoptotic Changes in the Aged Brain Are Triggered by Interleukin-1β-induced Activation of p38 and Reversed by Treatment with Eicosapentaenoic Acid

Among the several changes that occur in the aged brain is an increase in the concentration of the proinflammatory cytokine interleukin-1beta that is coupled with a deterioration in cell function. This study investigated the possibility that treatment with the polyunsaturated fatty acid eicosapentaenoic acid might prevent interleukin-1beta-induced deterioration in neuronal function. Assessment of four markers of apoptotic cell death, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and terminal dUTP nick-end staining, revealed an age-related increase in each of these measures, and the evidence presented indicates that treatment of aged rats with eicosapentaenoate reversed these changes as well as the accompanying increases in interleukin-1beta concentration and p38 activation. The data are consistent with the idea that activation of p38 plays a significant role in inducing the changes described since interleukin-1beta-induced activation of cytochrome c translocation and caspase-3 activation in cortical tissue in vitro were reversed by the p38 inhibitor SB203580. The age-related increases in interleukin-1beta concentration and p38 activation in cortex were mirrored by similar changes in hippocampus. These changes were coupled with an age-related deficit in long term potentiation in perforant path-granule cell synapses, while eicosapentaenoate treatment was associated with reversal of age-related changes in interleukin-1beta and p38 and with restoration of long term potentiation.

Donovanosis: an update.

Donovanosis has been ignored for many years until recently. The condition still has a limited geographical distribution. A significant epidemic of donovanosis has been identified in KwaZulu/Natal, South Africa where it may be a risk factor for acquiring HIV in men. After a gap of more than 30 years, the organism was cultured by researchers in Durban, South Africa and Darwin, Australia. Polymerase chain reaction (PCR) techniques for donovanosis were developed soon after, most recently using a colorimetric detection system. Similarities between the causative organism, Calymmatobacterium granulomatis and Klebsiella spp. were confirmed. A proposal that the organism be reclassified under the genus Klebsiella has been put forward. Azithromycin has been confirmed as the drug of choice but is yet to be accepted universally because of cost issues. Treatment in patients with significant HIV induced immune deficiency may need to be prolonged. A donovanosis eradication programme is underway amongst the aboriginal community in Australia. Elsewhere, management through current syndromic guidelines for genital ulcers are yet to be validated in areas where donovanosis is endemic. PCR testing should enable further recognition of donovanosis and lead to more concerted efforts in disease control and possible eradication.

A colorimetric detection system for Calymmatobacterium granulomatis

Objective: To incorporate the first polymerase chain reaction (PCR) assay for Calymmatobacterium granulomatis into a colorimetric detection system for use in routine diagnostic laboratories. Methods: A capture oligonucleotide specific for...

Development of a Quantitative Immunodipstick Assay for Phytophthora nicotianae

Monoclonal antibodies have been produced that are specific for the species, Phytophthora nicotianae, a plant pathogen which causes blackshank on tobacco and root rots in citrus. The antibodies react with the surface of P. nicotianae cysts but not with cysts of any other species of Phytophthora or Pythium tested. The antibodies have been used in a dipstick assay in which a colorimetric detection system gives a quantitative indication of the level of inoculum present. In this assay, one of the species-specific antibodies, Pn8E6, detected P. nicotianae spores at densities as low as 10 spores/ml. In preliminary tests, the assay was able to detect P. nicotianae in contaminated soil samples and the intensity of the reaction showed a correlation with results obtained from plating assays on selective media.

Early microbiologic diagnosis of spontaneous bacterial peritonitis with BacT/ALERT

Background/Aims: Inoculation of ascitic fluid into conventional blood culture bottles is more sensitive than conventional culture in the diagnosis of spontaneous bacterial peritonitis. BacT/ALERT is an automated colorimetric microbial detection system that has been shown to be faster than conventional blood culture bottles in the diagnosis of bacteremia. The aim of the study was to compare the BacT/ALERT system with the conventional culture and the conventional blood culture bottles method in the diagnosis of spontaneous bacterial peritonitis. Methods: All the ascitic fluid samples from patients with cirrhosis hospitalized in our Department between September 1992 and May 1994 ( n = 1032) were prospectively evaluated. In all cases, an aliquot of ascitic fluid was sent for Gram's stain and conventional culture, and 20 ml were inoculated at the bedside into blood culture bottles: 10 ml into conventional blood culture bottles and 10 ml into BacT/ALERT. Results: Thirty ascitic fluid infections (23 spontaneous bacterial peritonitis and 7 neutrocytic ascites) and 20 bacterascites were diagnosed. Conventional culture was positive in 10 30 ascitic fluid infections (33.3%), conventional blood culture bottles in 22 30 (73.3%) ( p<0.01 compared to conventional culture) and BacT/ALERT in 20 30 (66.6%) ( p<0.05 compared to conventional culture, pNS compared to conventional blood culture bottles). The time elapsed for culture positivity was 43.4±34.2 h for conventional blood culture bottles and 13.3±9.2 h for BacT/ALERT ( p<0.001). Thirteen of the 23 cases of spontaneous bacterial peritonitis (56.5%) were detected within the first 12 h with BacT/ALERT, as compared to only three (13%) with conventional blood culture bottles ( p<0.03). Conclusion: The automated system BacT/ALERT provides an earlier microbiologic diagnosis of spontaneous bacterial peritonitis than conventional blood culture bottles with similar sensitivity.

Detection of K-raspoint mutation by enriched PCR–colorimetric plate assay

Point mutations in the K-rasgene are frequently observed in a variety of human malignancies, including colorectal and pancreatic cancers. In this paper, we describe a sensitive procedure for the detection of point mutations of codon 12 of the K-rasgene. The assay employs a single-tube enriched PCR procedure, coupled to colorimetric detection. In the enriched PCR procedure, the first round of amplification introduces a restriction enzyme site in the wild type, but not in mutant K-rasPCR product. The wild type products are then digested and the second round of PCR enriches for the mutant sequences by amplifying the resistant products. The second round of amplification allows the incorporation of biotin and a substrate binding tag at opposite ends of the mutant product, thus allowing detection of the product by a simple colorimetric assay. The assay has been validated using DNA from a variety of cell lines known to contain either mutant or wild type K-ras. Under these conditions, the assay has proved both reproducible and sensitive, with the ability to detect one mutant molecule in a background of 1000 wild type molecules. The assay allowed discrimination of mutant from wild type K-rasin samples from colonic adenocarcinomas and normal colonic mucosa. The use of a colorimetric detection system reduces observer bias and facilitates analysis of large numbers of samples. As such, the assay may have specific application in the sensitive detection of K-rasmutations in a variety of clinical samples.

Specific Identification of Fusarium graminearum by PCR with gaoA Targeted Primers

Summary A duplex PCR procedure for the specific identification of Fusarium graminearum was set up and optimized for high sensitivity. The specific primer pair used was originally designed for the detection of a galactose oxidase producing F. graminearum -strain previously designated Cladobotryum dendroides NRRL 2903. Results obtained in this study suggest identification of this strain as F. graminearum . The PCR procedure gave a specific 900 bp amplificate with DNA isolated from F. graminearum (and from C. dendroides NRRL 2903). No amplification occurred with DNA from closely related Fusarium species, fungi of other genera, bacteria and cereals. The method was used for screening of 22 strains originally designated F. graminearum . Results were compared to morphological characteristics of the isolates, red pigmentation on mannitol-PCNB-agar, and production of galactose oxidase by a novel colorimetric detection system for this enzyme. Negative results in specific amplification with some of the isolates were in good accordance with physiological and morphological characterization. The value of this highly specific method for the detection of F. graminearum in natural habitats is discussed.

Volume of blood required to detect common neonatal pathogens

OBJECTIVE: To determine the minimum volume of blood and the absolute number of organisms required for detection of bacteremia and fungemia by an automated colorimetric blood culture system (BacT/Alert, Organon Teknika). DESIGN: Common neonatal pathogens, Escherichia coli, Streptococcus agalactiae (group B streptococcus [GBS]: one American Type Culture Collection [ATCC] strain and one clinical isolate), Staphylococcus epidermidis, and Candida albicans, were seeded into blood to produce bacteremia or fungemia with low colony counts (1 to 3 colony-forming units [CFU] per milliliter) and ultra-low colony counts (<1 CFU/ml). For each organism, 96 culture bottles were inoculated with either 0.25, 0.5, 1.0, or 4.0 ml of the two seeded blood concentrations. Blood culture bottles were incubated in the BacT/Alert device for 5 days, and time to positivity was noted when applicable. All bottles were subcultured on plated media. DATA ANALYSIS: The Poisson statistic was used to calculate the probability of finding at least one viable CFU per inoculated culture bottle. The fraction of culture bottles with positive findings per group was divided by the probability of one or more organisms present to give the positivity index. RESULTS: Plated subculture identified no growth of organisms not detected by the colorimetric detection system. The false-positive rate for the automated device was less than 1%. The positivity index for the GBS clinical isolate was 1.13, for the GBS ATCC isolate 0.96, for S. epidermidis 0.94, for C. albicans 0.97, and for E. coli 0.95. There was a statistically significant difference with time to positivity and inocula volume ( p <0.01), but the difference was not clinically important. CONCLUSIONS: If one or two viable colony-forming units are in the blood inoculated into culture media, the BacT/Alert system will detect growth rapidly. Because there appears to be a sizable subset of neonates who are at risk of sepsis with a colony count less than 4 CFU/ml, then a 0.5 ml inoculum of blood into the culture media is inadequate for sensitive and timely detection of bacteremia. One to two milliliters of blood should increase microorganism recovery in the face of low-colony-count sepsis. (J P EDIATR 1996;129:275-8)

Detection of Salmonella spp in estuarine waters by using both the conventional culture and a probe technique

The presence of Salmonella spp in estuarine waters was investigated along the Patras harbor where pipes containing urban sewage terminate. Salmonellae detection was performed by a conventional culture and a DNA probe technique (Gene-Trak Salmonella assay — Gene Trak Systems, Framingham). The Gene Trak colorimetric Salmonella assay uses the ribosomal hybridization format followed by a colorimetric detection system.