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At least 145 records · Page 8Linked to original sources

PubMed2004

Single nucleotide polymorphism analysis by chip-based hybridization and direct current electrical detection of gold-labeled DNA.

Single nucleotide polymorphism (SNP) analysis at the point of care requires a low cost detection technology that is capable of miniaturization, multiplexing, and high sensitivity. Direct current electrical detection (DCED) of DNA following nanoparticle labeling and silver enhancement is a promising candidate technology for point-of-care diagnostics. In this work we present, for the first time, SNP analysis in PCR products from patient samples using DCED, taking this platform technology a step closer to practical application. We developed a silane functionalized polymer for coating of biochip surfaces. This polymeric coating is stable under harsh conditions and has exceptionally high binding capacity. Allele-specific oligonucleotide probes were immobilized on chips coated with this polymer. Biotinylated PCR products of the human cholesteryl ester transfer protein gene from different patients were hybridized to the chips, labeled with gold nanoparticles, and autometallographically enhanced. The chips were scanned for DC electrical resistance by applying movable electrodes to the surface. Eighteen of nineteen patient samples were assigned the correct genotype. Our results demonstrate that SNP analysis of patient samples is feasible with DCED.

Affinity Labels↗

PubMed2002

Development of a facile fluorescent assay for the detection of 80 mutations within the p53 gene.

BACKGROUND: Alterations in the p53 tumor suppressor gene constitute one of the most frequent genetic events associated with the development of human cancers. Determination of an individual's p53 status may be of value in early diagnosis, prediction of response to treatment, and for the detection of minimal residual cancer. Recent studies have also revealed that specific mutations affecting the p53 gene are associated with a poor outcome. The majority of tumor biopsies that are sent for study in the laboratory contain neoplastic cells intermingled with stroma, such that the detection of alterations in the p53 gene requires a tumor enrichment technique and/or highly sensitive mutation detection technologies. Thus, it is desirable that a clinically useful assay for detecting point mutations in the p53 gene function in the presence of significant quantities of wild-type sequence and identify the critical sequence aberrations. MATERIALS AND METHODS: We utilized molecular beacons in a real-time allele-specific PCR format to obtain reference data on samples of quantitatively known p53 mutation status. These data have been statistically analyzed and the results used to detect p53 mutations, indicating the presence of occult tumor. RESULTS: We describe validation of a simple, rapid, sensitive, and quantitative ARMS assay for identifying the levels of 80 point mutations within the p53 gene that, when mutated, constitute at least 1% of the total p53 sequences. CONCLUSIONS: The assay successfully identifies rare p53 gene mutations in clinical samples and overcomes many of the limitations of current technologies.

Biological Assay↗

PubMed2001

Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.

A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

Animals↗

PubMed2003

Mutation analysis of the RET gene in total intestinal aganglionosis by wave DNA fragment analysis system.

BACKGROUND/PURPOSE: Total intestinal aganglionosis (TIA) extending from the duodenum to the rectum is the most rare form of Hirschprung's disease (HSCR) and usually is fatal. RET is the major gene associated with HSCR, and germline mutations of this gene account for up 50% of familial and up to 15 to 20% of sporadic cases in HSCR. The aim of this study was to investigate DNA variants in the RET gene in TIA patients using the WAVE DNA Fragment Analysis System. METHODS: Genomic DNA was extracted from whole blood samples from 6 patients with TIA. Polymerase chain reaction (PCR) amplification of the 21 exons of RET was performed using published oligonucleotide primers. Heteroduplexes were followed by the WAVE DNA Fragment Analysis System with the DNASep cartridge. RESULTS: WAVE system technology detected 16 variants in the RET gene in the 6 patients with TIA. Three patients had a significant mutation in exon 8, 11, and 15, respectively. Thirteen RET polymorphic variants also were detected in the 6 patients, with L746L variant in exon 13 occurring in 4 patients. CONCLUSIONS: WAVE system technology is an efficient method for the detection of DNA sequence variants. Our findings suggest that not only RET mutations but also RET polymorphic variants may contribute to the occurrence of TIA.

Amino Acid Substitution↗

PubMed2005

Use of a transgenic mouse model to identify markers of human lung tumors.

Lung cancer remains the leading cause of cancer related deaths worldwide. Despite advances in detection technologies, most patients diagnosed with lung cancer already harbor metastatic lesions. Because early detection is one of the primary determinants of patient outcome, a transgenic mouse model of lung cancer was utilized to identify markers of early lung tumors in humans. DNA microarray analysis of lung tumors arising in MMTV-IGF-II transgenic mice showed 9 genes consistently elevated in the murine lung tumors. Western blot analyses confirmed that several of these proteins were elevated in the lung tumors and immunohistochemical analyses identified 3 proteins, microsomal glutathione-S-transferase 1 (Mgst1), cathepsin H and syndecan 1 as being consistently elevated in the murine lung tumors compared to non-tumor bearing transgenic lung tissue and normal lung tissue surrounding the tumor. These 3 proteins were also elevated in human lung adenocarcinoma and squamous cell carcinomas. Importantly, the proteins were elevated in early stage, node negative tumors indicating their ability to detect early lung lesions that would be amenable to surgical resection. Therefore, our findings indicate that Mgst1, cathepsin H and syndecan 1 should be further evaluated as markers capable of identifying patients with early stage lung tumors.

Animals↗

PubMed2002

Development of a chemiluminescence-based ribonuclease protection assay.

The ribonuclease protection assay (RPA) is a widely used method for the detection and quantification of specific mRNA transcripts in a complex mixture of total RNA or mRNA molecules. While exhibiting many advantages over other RNA detection methods, RPAs are traditionally performed using radiolabeled probes that often require gel purification steps and lengthy exposure times to visualize results. Moreover, these probes can only be used for 1-2 weeks because of their short isotopic half-life and radiolysis. We report a method that improves the traditional RPA by replacing radiolabeled probes with biotinylated probes and lengthy exposure times with quick, streptavidin/HRP-based chemiluminescent detection technology. Biotinylated probes can be used without get purification and are stable for years, as opposed to weeks. Most importantly, our streptavidin/HRP-based chemiluminescent technology enables us to achieve sensitivity results similar to radioactive RPAs and to detect multiple transcripts in a single sample more efficiently. Furthermore, this new protocol addresses and eliminates the one major drawback unique to using biotinylated probes in chemiluminescent RPAs: a confounding artifact, not seen when running radioactive RPAs but commonly detected when using certain biotinylated rare message probes.

Actins↗

PubMed2004

Experiences in implementing uHTS--cutting edge technology meets the real world.

Driven by growing corporate compound files, the demands of target biology, and attempts to cut cost, the number of solutions to HTS has spiralled. In quick succession new assay technologies and screening platforms are appearing on the market, with the promise of screening faster than ever in low volume high density formats whilst providing high quality data. Within this world of rapid change, Pfizer has applied cutting edge technology to HTS by introducing screening in 1 microl formats utilising single molecule detection technology. Instead of resource intensive in-house development, Pfizer entered into a collaboration with Evotec OAI / Evotec Technologies and introduced their Mark-II EVOscreen platform. In this article we will outline the benefits of the approach taken at Pfizer, Sandwich, and introduce the Mark-II EVOscreen platform, illustrating the potential but also possible pitfalls of HTS miniaturisation.

Cloning, Molecular↗

PubMedDate not supplied

Dual-frequency ultrasound for detecting and sizing bubbles.

ISS construction and Mars exploration require extensive extravehicular activity (EVA), exposing crewmembers to increased decompression sickness risk. Improved bubble detection technologies could help increase EVA efficiency and safety. Creare Inc. has developed a bubble detection and sizing instrument using dual-frequency ultrasound. The device emits "pump" and "image" signals at two frequencies. The low-frequency pump signal causes an appropriately-sized bubble to resonate. When the image frequency hits a resonating bubble, mixing signals are returned at the sum and difference of the two frequencies. To test the feasibility of transcutaneous intravascular detection, intravascular bubbles in anesthetized swine were produced using agitated saline and decompression stress. Ultrasonic transducers on the chest provided the two frequencies. Mixing signals were detected transthoracically in the right atrium using both methods. A histogram of estimated bubble sizes could be constructed. Bubbles can be detected and sized transthoracically in the right atrium using dual-frequency ultrasound.

Animals↗

PubMed2004

Development and implementation of a highly miniaturized confocal 2D-FIDA-based high-throughput screening assay to search for active site modulators of the human heat shock protein 90beta.

The beta isoform of the heat shock protein 90 (Hsp90beta) is a cellular chaperone required for the maturation of key proteins involved in growth response to extracellular factors as well as oncogenic transformation of various cell types. Compounds that inhibit the function of Hsp90beta are thus believed to have potential as novel anticancer drugs. To date, 2 fungal metabolites are known to inhibit Hsp90beta. However, insolubility and liver toxicity restrict the clinical use of these molecules. The limitation to identify novel and safe Hsp90beta inhibitors is that presently no suitable high-throughput screening assay is available. Here, the authors present the development of a homogenous assay based on 2-dimensional fluorescence intensity distribution analysis of tetramethyl-rhodamine (TAMRA)-labeled radicicol bound to Hsp90beta. Furthermore, the assay has been shown to be compatible with the confocal nanoscreening platform Mark II from Evotec-Technologies and can therefore be used for miniaturized high-throughput screening. The applied detection technology provides critical information about the nature of biomolecular interaction at the thermodynamic equilibrium, such as affinity constants and stoichiometric parameters of the binding. The assay is used to identify small molecular weight compounds displacing TAMRA-radicicol. Such compounds are believed to be important molecules in the discovery of novel anticancer drugs.

Biological Assay↗

PubMed1997

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Chelating Agents↗

PubMed2002

Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening.

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.

Biological Assay↗

PubMed1996

Shelf life prediction: status and future possibilities.

Although there is rapid progress in the field of chemical detection technology, little of this technology appears to have found application in estimation of the remaining shelf life of foods and early detection of spoilage. Predictive microbiology aims to summarise the probable behaviour of specific spoilage organisms and the progression of spoilage processes in foods. The quantitative knowledge generated in the field of predictive microbiology provides a sound basis for the rational development of devices with which to monitor loss of product shelf life during storage, distribution and retail sale. To predict remaining shelf life accurately it is necessary, however, to consider the microbial ecology of the food system. Aspects of microbial ecology and physiology relevant to the spoilage of foods are briefly reviewed and the potential benefits of the use of predictive microbiology in shelf life estimation are described. These points are exemplified by reference to a modelling program undertaken to develop, validate and 'package' in an easily useable from, models of the effect of temperature, water activity and pH on the growth rate of psychrotrophic spoilage pseudomonads. Necessary properties of devices to monitor loss of shelf life are discussed. 'Bioindicators' are identified as potential monitors of spoilage and suggestions made for their development based on the concept of 'upper limiting bacterial growth' rates, for which preliminary evidence is presented.

Food Microbiology↗

PubMed2004

Bacterial separation and concentration from complex sample matrices: a review.

The use of many rapid detection technologies could be expanded if the bacteria were separated, concentrated, and purified from the sample matrix before detection. Specific advantages of bacterial concentration might include facilitating the detection of multiple bacterial strains; removal of matrix-associated assay inhibitors; and provision of adequate sample size reduction to allow for the use of representative food sample sizes and/or small media volumes. Furthermore, bacterial concentration could aid in improving sampling techniques needed to detect low levels of pathogens or sporadic contamination, which may perhaps reduce or even eliminate the need for cultural enrichment prior to detection. Although bacterial concentration methods such as centrifugation, filtration, and immunomagnetic separation have been reported for food systems, none of these is ideal and in many cases a technique optimized for one food system or microorganism is not readily adaptable to others. Indeed, the separation and subsequent concentration of bacterial cells from a food sample during sample preparation continues to be a stumbling block in the advancement of molecular methods for the detection of foodborne pathogens. The purpose of this review is to provide a detailed understanding of the science, possibilities, and limitations of separating and concentrating bacterial cells from the food matrix in an effort to further improve our ability to harness molecular methods for the rapid detection of foodborne pathogens.

Bacteria↗

PubMed2004

Immunoassays based on electrochemical detection using microelectrode arrays.

We show that CombiMatrix's VLSI arrays of individually addressable electrodes, using conventional CMOS integrated circuitry, can be used in detecting various analytes via immunoassay protocols. These microarrays provide over 1000 electrodes per square centimeter. The chips are coated with a porous material on which specific affinity tags are synthesized proximate to selected electrode sites. CombiMatrix microarrays are used to develop spatially multiplexed assay formats for biological entities over a wide range of sizes, from small molecules to cells. Antibodies are tagged with coded affinity labels and then allowed to self-assemble on the appropriate electrode assay sites. Each analyte-specific antibody is chaperoned to individual, predetermined locations by the self-assembly process. The resulting chip can perform numerous different analyte-specific immunoassays, simultaneously. We present new detection technologies based upon the use of the active individually addressable microelectrodes on the chip: redox enzyme amplified electrochemical detection. The results for human alpha1 acid glycoprotein, ricin, M13 phage, Bacillus globigii spores, and fluorescein indicate that this method is one of the most sensitive available, with limits of detection in the attomole range. The detection range is 4-5 logs of analyte concentration, with an assay volume of 50 microl or less. The system provides for a host of multiplexed immunoassays because of the large number of electrodes available. We show how the assays can be optimized for maximum performance on the CombiMatrix microarray platform.

Biosensing Techniques↗

PubMed2005

[Laboratory methods for detection and identification of biological pathogens].

Laboratory detection and recognition methods of infectious diseases agents have developed markedly in recent years, following the proliferation of nucleic acid and immuno-based detection technologies. The present review summarizes the state of the art in current biorecognition methods: antigenic identification, genetic identification such as PCR, RFLP and FISH, protemics and mass spectrometry. For each method we have specified the technology and qualification required, time to result, specifity and sensitivity, while emphasizing the advantages and disadvantages of using each method for the detection of a given pathogen. Nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is simpler and expected to further development with the improvements in the affinity, specifity and mass production of new immunoglobulins. Protein-based detection methods have an advantage comparing to nucleic acid identification: the presence of the protein approves that the tested gene is functional. Mass spectrometry enables simultaneous detections of multiple proteins and thus holds a promise for new technical developments with a vast array of applications. Most physicians do not practice biodetection technologies in their every day routine, but encounter those terms in their clinical and academic work. The review aims to display basic information in this field in order to enable a common language with basic science specialists.

Bacteria↗

PubMed2005

A review of technologies for rapid detection of bacteria in recreational waters.

Monitoring of recreational beaches for fecal indicator bacteria is currently performed using culture-based technology that can require more than a day for laboratory analysis, during which time swimmers are at risk. Here we review new methods that have the potential to reduce the measurement period to less than an hour. These methods generally involve two steps. The first is target capture, in which the microbial group of interest (or some molecular/chemical/or biochemical signature of the group) is removed, tagged or amplified to differentiate it from the remaining material in the sample. We discuss three classes of capture methods: 1) Surface and whole-cell recognition methods, including immunoassay techniques and molecule-specific probes; 2) Nucleic acid methods, including polymerase chain reaction (PCR), quantitative PCR (Q-PCR), nucleic acid sequence based amplification (NASBA) and microarrays; and 3) Enzyme/substrate methods utilizing chromogenic or fluorogenic substrates. The second step is detection, in which optical, electrochemical or piezoelectric technologies are used to quantify the captured, tagged or amplified material. The biggest technological hurdle for all of these methods is sensitivity, as EPA's recommended bathing water standard is less than one cell per ml and most detection technologies measure sample volumes less than 1 ml. This challenge is being overcome through addition of preconcentration or enrichment steps, which have the potential to boost sensitivity without the need to develop new detector technology. The second hurdle is demonstrating a relationship to health risk, since most new methods are based on measuring cell structure without assessing viability and may not relate to current water quality standards that were developed in epidemiology studies using culture-based methods. Enzyme/substrate methods may be the first rapid methods adopted because they are based on the same capture technology as currently-approved EPA methods and their relationship to health risk can be established by demonstrating equivalency to existing procedures. Demonstration of equivalency may also be possible for some surface and whole-cell recognition methods that capture bacteria in a potentially viable state. Nucleic acid technologies are the most versatile, but measure nonviable structure and will require inclusion in epidemiological studies to link their measurement with health risk.

Bacteria↗

PubMed2025

Sensitive and Visualized Detection of Hantavirus Using CRISPR/Cas12a Based on AutoCORDSv2 Design.

In recent years, detection technologies based on the CRISPR/Cas12a method have been extensively utilized in the fields of nucleic acid, enzyme, and macromolecule detection, thereby reinforcing their significant role in the detection landscape. Enhancing the simplicity of design, efficiency, and automation of the CRISPR/Cas12a detection system is essential for advancing its application in diagnostics. Recently, we developed an automated CRISPR/Cas12a design system named AutoCORDSv2. This system can process published genomic sequences of pathogenic bacteria in a high-throughput manner and automatically generate conserved and highly specific crRNA sequences, along with primer sequences for target amplification. This capability facilitates the specific and precise design of the CRISPR/Cas12a detection system. In this study, crRNAs targeting the Hantaan virus (HTNV) and Seoul virus (SEOV), as well as RT-PCR primers and RT-RPA primers, were designed using AutoCORDSv2. The experimental results demonstrated that the CRISPR/Cas12a system, automatically designed by AutoCORDSv2, was specific for the detection of both the HTNV and SEOV, with no cross-reactivity observed with other pathogens. The detection sensitivity reached 6 copies/μL (equivalent to 111 copies per amplification reaction), whether measured by a microplate reader or directly observed with the naked eye. The detection results for 50 samples were consistent with those obtained from commercial RT-qPCR kits, indicating high precision. Furthermore, the CRISPR/Cas12a system designed by AutoCORDSv2 can also be utilized for the development of a single-tube detection system with a sensitivity of 42 copies per reaction. This system combined with a 5-min extraction step and RT-RPA, further underscoring its potential for application.

CRISPR-Cas Systems↗

PubMed2003

Modeling of DNA hybridization kinetics for spatially resolved biochips.

The marriage of microfluidics with detection technologies that rely on highly selective nucleic acid hybridization will provide improvements in bioanalytical methods for purposes such as detection of pathogens or mutations and drug screening. The capability to deliver samples in a controlled manner across a two-dimensional hybridization detection platform represents a substantial technical challenge in the development of quantitative and reusable biochips. General theoretical and numerical models of heterogeneous hybridization kinetics are required in order to design and optimize such biochips and to develop a quantitative method for online interpretation of experimental results. In this work we propose a general kinetic model of heterogeneous hybridization and develop a technique for estimating the kinetic coefficients for the case of well-spaced, noninteracting surface-bound probes. The experimentally verified model is then incorporated into the BLOCS (biolab-on-a-chip simulation) 3D microfluidics finite element code and used to model the dynamic hybridization on a biochip surface in the presence of a temperature gradient. These simulations demonstrate how such a device can be used to discriminate between fully complementary and single-base-pair mismatched hybridization using fluorescence detection by interpretation of the unique spatially resolved intensity pattern. It is also shown how the dynamic transport of the targets is likely to affect the rate and location of hybridization as well as that, although nonspecific hybridization is present, the change in the concentration of hybridized targets over the sensor platform is sufficiently high to determine if a fully complementary match is present. Practical design information such as the optimum transport speed, target concentration, and channel height is presented. The results presented here will aid in the interpretation of results obtained with such a temperature-gradient biochip.

Biosensing Techniques↗