Simultaneous Analysis Of Multiple Samples Research Articles

Evaluation of 4-fluoro-7-nitrobenzofurazan as a dual-function chromogenic and fluorogenic probe for tulathromycin and its innovative utility for development of two eco-friendly and high-through microwell assays for analysis of pharmaceutical formulations

Tulathromycin is a triamilide antibacterial drug which has been approved for use in the European Union and the United States for the treatment and prevention of bovine respiratory diseases. The aim of this study was the development of two innovative microwell spectrometric (photometric and fluorometric) assays for determination of tulathromycin in its pharmaceutical formulations. To achieve this goal, 4-fluoro-7-nitrobenzofurazan was investigated as a dual-function chromogenic and fluorogenic probe for tulathromycin. The reaction between tulathromycin and 4-fluoro-7-nitrobenzofurazan proceeded smoothly in an alkaline medium, resulting in the formation of a colored and fluorescent product. The product displayed a maximum light absorption at 475 nm and emitted fluorescence at 545 nm when excited at 475 nm. Extensive investigations were conducted to optimize the factors affecting the tulathromycin /4-fluoro-7-nitrobenzofurazan reaction, and the optimal conditions were established. Using these optimized conditions, both microwell-based photometric and fluorometric assays were developed. The calibration curves relating the absorbance and fluorescence intensities of the reaction product with the corresponding tulathromycin concentrations were generated. The absorbance-concentration relationship was found to be linear within a tulathromycin concentration range of 10–400 µg mL−1, with a limit of quantitation of 6.2 µg mL−1. On the other hand, the fluorescence-concentration relationship was linear within a concentration range of 0.04–1.2 µg mL−1, with a limit of quantitation of 0.06 µg mL−1. Rigorous validations of both assays’ procedures were performed, and both assays were successfully employed for the analysis of tulathromycin–containing pharmaceutical formulations (injections) with satisfactory accuracy and precision. The ecologically friendly assessment of both assays demonstrated their compliance with the principles of green analytical chemistry approaches. Moreover, the proposed microwell-based assays enabled the simultaneous analysis of multiple samples using small volumes, enabling high-throughput analysis. In conclusion, this study represents the first evaluation of 4-fluoro-7-nitrobenzofurazan as a probe with dual functionality for the microwell-based photometric and fluorometric analysis of tulathromycin. The developed assays serve as valuable analytical tools for ensuring the quality of tulathromycin ’s pharmaceutical formulations.

Weak Value Amplification Based Optical Sensor for High Throughput Real-Time Immunoassay of SARS-CoV-2 Spike Protein.

The demand for accurate and efficient immunoassays calls for the development of precise, high-throughput analysis methods. This paper introduces a novel approach utilizing a weak measurement interface sensor for immunoassays, offering a solution for high throughput analysis. Weak measurement is a precise quantum measurement method that amplifies the weak value of a system in the weak interaction through appropriate pre- and post-selection states. To facilitate the simultaneous analysis of multiple samples, we have developed a chip with six flow channels capable of conducting six immunoassays concurrently. We can perform real-time immunoassay to determine the binding characteristics of spike protein and antibody through real-time analysis of the flow channel images and calculating the relative intensity. The proposed method boasts a simple structure, eliminating the need for intricate nano processes. The spike protein concentration and relative intensity curve were fitted using the Log-Log fitting regression equation, and R2 was 0.91. Utilizing a pre-transformation approach to account for slight variations in detection sensitivity across different flow channels, the present method achieves an impressive limit of detection(LOD) of 0.85 ng/mL for the SARS-CoV-2 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, with a system standard deviation of 5.61. Furthermore, this method has been successfully verified for monitoring molecular-specific binding processes and differentiating binding capacities.

PSIV-20 An in Vitro Digestion Procedure Using Daisyii Incubator Can Replace Conventional in Vitro Digestion Procedures Using Flasks for Estimating Ileal Digestibility of Feed Ingredients for Pigs

Abstract In vitro procedures using flasks have been used for simulating nutrient utilization by pigs. However, these procedures are time-consuming and laborious and require a number of flasks and filter crucibles. The objective of this study was to develop an in vitro procedure using DaisyII incubator for simultaneous analysis of multiple samples. In a conventional in vitro procedure, 10 mg of pepsin in step 1 and 50 mg of pancreatin in step 2 are used for each flask containing 1 g of feed sample. In a preliminary experiment, 24 samples each in R55 bags were digested in a jar of DaisyII incubator with 24 times the quantity of buffer solution and enzymes of the conventional flask procedure. In the preliminary experiment, greater crude protein (CP) digestibility values were observed in the DaisyII incubator procedure compared with the conventional flask procedure. To determine optimum enzyme doses in the in vitro ileal disappearance (IVIDDaisy) procedure using the DaisyII incubator, 3 doses of pepsin (10, 7.5, and 5 mg/sample in step 1) and 3 doses of pancreatin (50, 37.5, and 25 mg/sample in step 2) were tested in a 3 × 3 factorial arrangement for 11 test ingredients. The quantity of buffer solution and enzymes in a jar was increased proportionally with the number of samples. The 11 test ingredients were barley, copra meal, corn, corn distillers dried grains with solubles, corn gluten feed, corn gluten meal, palm kernel meal, rice bran, soybean hulls, soybean meal, and wheat bran. The in vitro ileal disappearance (IVIDflask) of CP in the 11 ingredients was determined using the conventional flask procedure. The sum of square of deviation between IVIDflask of CP and IVIDDaisy of CP was the least when 5 mg pepsin/sample in step 1 and 37.5 mg/sample in step 2 were used (IVIDflask of CP, % = 14.4 + 0.81 × IVIDDaisy of CP, %; r2 = 0.748). On the other hand, the determination coefficient was the greatest when 10 mg pepsin/sample in step 1 and 50 mg/sample in step 2 were used (IVIDflask of CP, % = 2.5 + 0.93 × IVIDDaisy of CP, %; r2 = 0.878). In conclusion, the in vitro procedure using a DaisyII incubator can be used for estimating ileal digestibility of protein in multiple samples simultaneously for pigs.

Aptamer-based LoC-SERS sensing system for rapid and highly sensitive detection of gastric cancer

Multiplex detection of biomarkers is nontrivial for gastric cancer diagnosis. Herein, we presented a lab-on-a-chip surface-enhanced Raman scattering (LoC-SERS) sensing system for the detection of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B). The sensing system includes three parallel channels, each channel consists of two detection chambers, enabling the simultaneous analysis of multiple samples. The capillary structure provides self-driving power without the need of external pumps. The ordered gold nano-bipyramids (GNBPs) array in the detection chamber can form uniform and dense “hot spots”, due to the homogeneous double-tip structure and narrow gaps, resulting in better SERS performance. During the analysis process, VEGF and PDGF-B in the serum can lead to the change of aptamer conformation. Raman reporter-labeled complementary DNA is shed from the aptamer due to competition of the target. The entire process only requires 20 min, which is rapid. By monitoring the changes in Raman intensity and fitting it with the logarithm of concentration, the correlation between the intensity and concentration can be clearly reflected as a mathematical relationship. The LoC-SERS sensing system has been verified to have excellent selectivity and reproducibility. Via the sensing system, the limits of detection (LOD) of VEGF and PDGF-B are as low as 0.342 pg/mL and 0.265 pg/mL in serum, respectively. In addition, the accuracy was verified through enzyme-linked immunosorbent assay (ELISA). Therefore, the proposed LoC-SERS sensing system has great potential for clinical detection of gastric cancer.

A pump-free and high-throughput microfluidic chip for highly sensitive SERS assay of gastric cancer-related circulating tumor DNA via a cascade signal amplification strategy

Circulating tumour DNA (ctDNA) has emerged as an ideal biomarker for the early diagnosis and prognosis of gastric cancer (GC). In this work, a pump-free, high-throughput microfluidic chip coupled with catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) as the signal cascade amplification strategy (CHA–HCR) was developed for surface-enhanced Raman scattering (SERS) assays of PIK3CA E542K and TP53 (two GC-related ctDNAs). The chip consisted of six parallel functional units, enabling the simultaneous analysis of multiple samples. The pump-free design and hydrophilic treatment with polyethylene glycol (PEG) realized the automatic flow of reaction solutions in microchannels, eliminating the dependence on external heavy-duty pumps and significantly improving portability. In the reaction region of the chip, products generated by target-triggered CHA initiated the HCR, forming long nicked double-stranded DNA (dsDNA) on the Au nanobowl (AuNB) array surface, to which numerous SERS probes (Raman reporters and hairpin DNA-modified Cu2O octahedra) were attached. This CHA–HCR strategy generated numerous active “hot spots” around the Cu2O octahedra and AuNB surface, significantly enhancing the SERS signal intensity. Using this chip, an ultralow limit of detection (LOD) for PIK3CA E542K (1.26 aM) and TP53 (2.04 aM) was achieved, and the whole process was completed within 13 min. Finally, a tumour-bearing mouse model was established, and ctDNA levels in mouse serum at different stages were determined. To verify the experimental accuracy, the gold-standard qRT–PCR assay was utilized, and the results showed a high degree of consistency. Thus, this rapid, sensitive and cost-effective SERS microfluidic chip has potential as an ideal detection platform for ctDNA monitoring.

Design of a high-throughput bio-ferrograph for isolation of cancer cells from whole blood.

Enumeration and morphological characterization of circulating tumor cells (CTCs) can be useful in diagnosis and prognosis of metastatic cancer patients. The bio-ferrograph (BF) with its five flow channels, which was developed in the late 1990s for magnetic isolation of biological cells and tissue fragments from fluids, is a modification of the analytical ferrograph. Its use for isolation of rare CTCs from human whole blood (HWB) is a novel approach for the detection of cancer at a cellular level. The isolation process is facilitated by the interaction of specifically magnetized cells with a strong external magnetic field, yielding high recovery rates with no morphological alternation of cells that are isolated on a coverslip glass slide, thus allowing complementary microscopic, chemical, biological, and mechanical analyses. Here, a full mechanical and magnetostatic design of a novel high-throughput BF is presented. The system design is based on an optimized procedure for bio-ferrographic isolation of CTCs from HWB. It incorporates a semi-automated CTC separation system consisting of sample preparation, labeling, and staining; magnetic isolation; and system recovery. The design process was optimized based on experimental feasibility tests and finite element analyses. The novel bench-top system consists of 100 flow channels, allowing simultaneous analysis of multiple samples from 20 patients in each run, with the potential to become a decision-making tool for medical doctors when monitoring patients in a hospital setting. It opens a new route for early diagnosis, prognosis, and treatment of cancers, as well as other diseases, such as osteoarthritis.

Anchoring zinc-doped carbon dots on a paper-based chip for highly sensitive fluorescence detection of copper ions.

In this work, zinc-doped carbon dots (Zn-CDs) were anchored on a three-dimensional wheel type paper-based microfluidic chip, and were decorated with 6-mercaptonicotinic acid (MNA) and L-cysteine (L-Cys) for highly sensitive and rapid fluorescence detection of Cu2+. Zn-CDs were first anchored on paper through the amide bonds between the carboxyl groups of the Zn-CDs and the amino groups of the paper. Afterwards, Zn-CDs were decorated with MNA and L-Cys, effectively preventing the Zn-CDs from aggregation. The nitrogen atom on the pyridine ring and the carboxylic acid groups in MNA and L-Cys coordinated with Cu2+ to form a nonfluorescent ground-state complex, causing the fluorescence quenching of the Zn-CDs. The three-dimensional rotary design could simplify the operation process and achieve simultaneous analysis of multiple samples with different concentrations. Under optimal conditions, the fluorescent sensor exhibits linear response for the determination of Cu2+ in the range from 0.1 to 60 μg L-1 with the detection limit (LOD) of 0.018 μg L-1. The proposed strategy provides a novel way for the highly sensitive detection of Cu2+ in a complex water environment.

An innovative diagnostic strategy for the detection of rare molecular targets to select cancer patients for tumor-agnostic treatments.

Targeted therapies are playing an increasing role in oncology. Among them, particular attention is nowadays reserved to histology-agnostic treatments. Rare molecular alterations affecting different neoplastic forms, such as Microsatellite Instability (MSI), Neurotropic Tyrosine Receptor Kinase (NTRK) gene fusions, etc., can allow efficient treatments, irrespective of the histologic type. Developing an effective testing strategy for the detection of rare molecular alterations is challenging.We report an innovative diagnostic strategy for a rapid and economically affordable detection of this uncommon targets. Malignant tumor samples are selected at the time of histopathological diagnosis and further processed for simultaneous analysis of multiple samples on Tissue Micro Arrays (TMAs) and Tissue Slice Arrays (TSAs). The TSA approach was specifically designed for large scale screening of small biopsies. TMA sections and TSA were first screened by immunohistochemistry (IHC) for the expression of mismatch repair and TRK proteins. Positive cases were subjected to confirmation tests (fragment analysis/FISH/NGS).In a series of 1865 malignant tumors, 48 (2.6%) MSI cases and 6 (0.3%) NTRK fusion cases were detected in 9 and 4 different tumor forms, respectively. On average, the TMA/TSA screening approach enabled IHC analysis of about 20 patients simultaneously with significant saving of time and costs. In addition, we have shown that multiplex IHC can further increment the throughput. A detailed procedure for application of this diagnostic approach in clinical practice is reported.The strategy described may allow an efficient and sustainable selection of tumors carrying rare molecular targets, not to leave behind patients for effective agnostic treatments.

Development of a biosensing platform based on a laccase-hydrophobin chimera.

A simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.

Joint approximate diagonalization of eigenmatrices as a high-throughput approach for analysis of hyphenated and comprehensive two-dimensional gas chromatographic data

The objective of the present work is development of joint approximate diagonalization of eigenmatrices (JADE) as a member of independent component analysis (ICA) family, for the analysis of gas chromatography-mass spectrometry (GC–MS) and comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC–MS) data to address incomplete separation problem occurred during the analysis of complex sample matrices. In this regard, simulated GC–MS and GC×GC–MS data sets with different number of components, different degree of overlap and noise were evaluated. In the case of simultaneous analysis of multiple samples, column-wise augmentation for GC–MS and column-wise super-augmentation for GC×GC–MS was used before JADE analysis. The performance of JADE was evaluated in terms of statistical parameters of lack of fit (LOF), mutual information (MI) and Amari index as well as analytical figures of merit (AFOMs) obtained from calibration curves. In addition, the area of feasible solutions (AFSs) was calculated by two different approaches of MCR-BANDs and polygon inflation algorithm (FACPACK). Furthermore, JADE performance was compared with multivariate curve resolution-alternating least squares (MCR-ALS) and other ICA algorithms of mean-field ICA (MFICA) and mutual information least dependent component analysis (MILCA). In all cases, JADE could successfully resolve the elution and spectral profiles in GC–MS and GC×GC–MS data with acceptable statistical and calibration parameters and their solutions were in AFSs. To check the applicability of JADE in real cases, JADE was used for resolution and quantification of phenanthrene and anthracene in aromatic fraction of heavy fuel oil (HFO) analyzed by GC×GC–MS. Surprisingly, pure elution and spectral profiles of target compounds were properly resolved in the presence of baseline and interferences using JADE. Once more, the performance of JADE was compared with MCR-ALS in real case. On this matter, the mutual information (MI) values were 1.01 and 1.13 for resolved profiles by JADE and MCR-ALS, respectively. In addition, LOD values (μg/mL) were respectively 1.36 and 1.24 for phenanthrene and 1.26 and 1.09 for anthracene using MCR-ALS and JADE which showed outperformance of JADE over MCR-ALS.

Fast and accurate mutation detection in whole genome sequences of multiple isogenic samples with IsoMut

BackgroundDetection of somatic mutations is one of the main goals of next generation DNA sequencing. A wide range of experimental systems are available for the study of spontaneous or environmentally induced mutagenic processes. However, most of the routinely used mutation calling algorithms are not optimised for the simultaneous analysis of multiple samples, or for non-human experimental model systems with no reliable databases of common genetic variations. Most standard tools either require numerous in-house post filtering steps with scarce documentation or take an unpractically long time to run. To overcome these problems, we designed the streamlined IsoMut tool which can be readily adapted to experimental scenarios where the goal is the identification of experimentally induced mutations in multiple isogenic samples.MethodsUsing 30 isogenic samples, reliable cohorts of validated mutations were created for testing purposes. Optimal values of the filtering parameters of IsoMut were determined in a thorough and strict optimization procedure based on these test sets.ResultsWe show that IsoMut, when tuned correctly, decreases the false positive rate compared to conventional tools in a 30 sample experimental setup; and detects not only single nucleotide variations, but short insertions and deletions as well. IsoMut can also be run more than a hundred times faster than the most precise state of art tool, due its straightforward and easily understandable filtering algorithm.ConclusionsIsoMut has already been successfully applied in multiple recent studies to find unique, treatment induced mutations in sets of isogenic samples with very low false positive rates. These types of studies provide an important contribution to determining the mutagenic effect of environmental agents or genetic defects, and IsoMut turned out to be an invaluable tool in the analysis of such data.

Rapid and Semi-Automated Screening of Multiple Samples for Antigen-Specific T Lymphocytes

Abstract 4839Functional characterizations of T lymphocytes are performed to gain understanding on their contribution in certain immunological situations, to monitor the course of diseases, and to track therapeutic interventions. Meanwhile the flow cytometric analysis of antigen-specific T cells examined for intracellular cytokine production and expression of activation markers after a short-term in vitro antigenic challenge is a well-established method for research applications. Despite the advantages of this approach to qualify samples on a single cell level and by multiple parameters, the broad use of this analysis for immune monitoring purposes is hampered. Screening of a lot of samples is time-consuming, requires many manual handling steps, and operator experience in flow cytometric analysis of stimulated T cell samples. To overcome these hurdles, we worked out a complete strategy to rapidly study cytokine and activation marker profiles in antigen-specific T cells of multiple samples by a semi-automated process.For the simultaneous analysis of multiple samples we examined for the T cell stimulation an antigen pre-coated 96-strip-well culture system. This flexible and ready-to-use format provides the opportunity to screen either for a single antigen or in parallel for up to twelve antigen specificities by combining 8-well-strips possessing different antigens. The coated antigens consist of pools of overlapping 15-mer peptides derived from a single viral protein of cytomegalovirus, Epstein-Barr-Virus, or adenovirus. The peptide pools have been designed for activation of the specific CD4+ as well as CD8+ T cells. They are solubilized and thereby accessible for T cell stimulation after addition of a cell sample, e.g. peripheral blood mononuclear cells, suspended in culture medium into the antigen-coated well. After a stimulation period of six hours the induced T cell response is comparable to the activation with a conventional lyophilized and reconstituted peptide pool. To reduce the time and work load for cell harvesting, fixation, permeabilization, and staining, we developed a protocol and reagents to allow a rapid and easy-to-handle intracellular staining procedure. Compared to conventional staining protocols, all steps are executed in the 96-strip-well culture plate, i.e. cell harvesting is dispensable. Without any washing step, cells are fixed and stained with defined reagent cocktails containing antibodies to identify virus-specific CD3+ CD4+ CD154+ and/or CD3+ CD8+ T cells and various Anti-cytokine-fluorochrome conjugates to evaluate the cytokine pattern. With these modifications, we drastically diminished the overall processing time for the staining of up to 96 samples to only 50 minutes. Furthermore, we integrated an automated flow cytometric analysis process. This includes the possibility to measure the samples in the 96-strip-well plates hands-free using pre-defined experiment settings and acquisition templates. We also applied an automated gating strategy for the data analysis. Finally, a report summarizes the results of the T cell response against several viral proteins for all samples tested, e.g. frequencies of cytokine+ CD154+ CD4+ and cytokine+ CD8+ T cell subsets are indicated. Hands-on time for the multi-sample acquisition and analysis is only minimal and the standardized reagents/protocol and sample analysis process decrease inter- and intra-assay variations.In summary, with our newly developed tools and protocols for in vitro T cell stimulation, staining of activation markers as well as intracellular cytokines, and automated flow cytometric analysis we have set up a fast and convenient procedure to routinely monitor antigen-specific T cell responses. Disclosures:Richter:Miltenyi Biotec GmbH: Employment. Niemöller:Miltenyi Biotec GmbH: Employment. Verwohl:Miltenyi Biotec GmbH: Employment. Lange:Miltenyi Biotec GmbH: Employment. Foerster-Marniok:Miltenyi Biotec GmbH: Employment. Brauns:Miltenyi Biotec GmbH: Employment. Kramer:Miltenyi Biotec GmbH: Employment. Höher-Peters:Miltenyi Biotec GmbH: Employment. Büscher:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment.

Use of denaturing gradient gel electrophoresis in screening unknown β-thalassemia mutations in Egyptian patients

The molecular defects resulting in a β-thalassemia phenotype, in the Egyptian population show a clear heterogenic pattern. Many studies have embarked on the molecular detection and characterization of these mutations, using a wide array of the available techniques with successful detection of both known and unknown mutations. PCR based techniques, as well as direct DNA sequencing are effective with some limitations as regards the time, effort and high cost to reach a final diagnosis. Intermediary screening techniques have proved to be effective tools to overcome these drawbacks. This study aims to assess the use of the denaturing gradient gel electrophoresis (DGGE)1DGGE1: denaturing gradient gel electrophoresis.1 to detect β-thalassemia mutations prior to the performance of direct sequencing to minimize the cost and workload involved in the process. In this study, forty-two previously genotyped patients in a study by El-Gawhary et al. in 2007, have been analyzed by DGGE for fragment 2 then 1. These are the β-globin gene fragments showing the majority of the β-thalassemia mutations. Sixty-eight alleles out of 79 mutant alleles in total were detected within these two fragments. The 11 undetected alleles comprise 9 alleles that require further examination using other DGGE fragments (0, 4 and 5) and correspond to -87(C>G), intervening sequence (IVS)II-1(G>A), IVS II-745, and IVS II-848(C>A). The remaining two that failed detection correspond to codon (CD) 37(G>A). Although, IVS-II 745(C>G) is undetectable within these two fragments, its 100% linkage polymorphism (+20 C>T) was detected in fragment 1 gel. DGGE is a sensitive technique to screen for β-thalassemia mutations. For simultaneous analysis of multiple samples with unknown mutations, it is recommended that direct DNA sequencing be coupled with DGGE whenever available to reduce time, effort and cost.

Simultaneous determination of biomarkers for Alzheimer's disease using sol–gel-derived optical array biosensor

The sol–gel-derived optical array-based biosensor for the simultaneous analysis of multiple analytes as potential markers for Alzheimer's disease was fabricated. β-Amyloid, acetylcholine and glutamate were selected as the biosensing probes. The fluorescent dye, carboxy SNARF-1-dextran, was co-immobilized with glutamate dehydrogenase and acetylcholinesterase for sensing glutamate and acetylcholine, respectively, while Amplex red and FITC-dextran were immobilized with horseradish peroxidase for determination of β-amyloid and hydrogen peroxide (H 2O 2). The biosensors exhibited a good performance on simultaneous analysis of multianalytes without obvious cross-interference and the detection limits of H 2O 2, β-amyloid, glutamate and acetylcholine were 1.57 nM, 0.63 nM, 0.55 μM, and 1.0 μM, respectively. The developed array biosensors were also applied to determine multianalytes in human serum samples spiked with various concentrations of analytes and showed a good analytical performance with dynamic range of 4 orders of magnitude for β-amyloid. Results obtained in this study clearly demonstrate the possibility of using a sol–gel-derived optical array-based biosensor for simultaneous analysis of multiple samples in the presence of important analytes for Alzheimer's disease.

Design and construction of a micromilled fluidic device as part of a DNA biosensor

Under the Optonanogen project (EU contract IST-2001-37239), a novel biosensor has been developed, which incorporates a disposable acrylic polymethylmethacrylate (PMMA) fluidic header. This header is designed to deliver a sample to a series of chemically primed cantilevers where hybridization of target DNA sequences and resulting deflection of the cantilevers is detected optically. Two different microfluidic headers are described, which are designed to incorporate the cantilever chip and which demonstrate a novel approach to microfluidic header assembly, integration with macroscale fluidics, fluidic handling, and priming strategies. The first header facilitates the delivery of a single fluid sample to all cantilevers, whereas the second permits discrete delivery of samples to isolated cantilevers, despite all cantilevers being contained on a single chip. This second, multi-path header therefore allows simultaneous analysis of multiple samples, or multiple parallel tests on a single sample. This paper describes these headers and for the multi-path device details the design changes incorporated to ensure effective isolation of the sample including a novel valve to improve priming of the microfluidic circuit.

Novel miniature SPR immunosensor equipped with all-in-one multi-microchannel sensor chip for detecting low-molecular-weight analytes

A simple and versatile miniaturized surface plasmon resonance (SPR) immunosensor enabling parallel analysis of multiple analytes or multiple samples of an analyte has been investigated for detection of a low-molecular-weight (lmw) toxin, 2,4-dichlorophenoxyacetic acid (2,4-D). A specially designed multi-microchannel SPR sensor module, integrating an optical-prism coated with an array of thin Au-films, a multi-microchannel plate (eight channels) and a flow-cell together, has been fabricated. The sensing surface was fabricated simply by physical adsorption of a protein conjugate of 2,4-D, and an indirect competitive immunoassay principle has been applied for the quantification of 2,4-D. Multiple 2,4-D samples were analyzed in a single step and a low-detection-limit (LDL) of 0.1 ppb (ng ml −1) 2,4-D was established. Competence of the portable SPR immunosensor for selective detection of 2,4-D despite the presence of various structurally resemblant interferents and from river-water samples has been demonstrated. The independent all-in-one sensor module highly favors shelf-storage between multiple determinations, and reusability of a same multi-microchannel flow-module for more than 35 days with intermittent storage (4–8 °C) has been confirmed. The LDL of 2,4-D could be enhanced further by introducing a simple avidin–biotin interaction-based sandwich immunoassay, with which the sensor signal multiplied enormously by a factor of ca. 10 and the LDL enhanced to 0.008 ppb. The miniature SPR sensor demonstrated here for simultaneous analysis of multiple samples with reusability and good storage ability is an important consideration for the advancement of biosensor technology.

TRAMPR: AN R package for analysis and matching of terminal‐restriction fragment length polymorphism (TRFLP) profiles

Abstracttrampr (TRFLP analysis and matching package for r) is a package for matching multiple terminal restriction fragment length polymorphism (TRFLP) profiles between unknown samples and a database of known TRFLP profiles in order to infer the presence of species in environmental samples. It permits simultaneous analysis of multiple samples and facilitates direct workflow from electrophoresis output through to community analyses. trampr also resolves the issues of multiple TRFLP profiles within a species and (conversely) shared TRFLP profiles across species.

Aberrant DNA Methylation of a Cell Cycle Regulatory Pathway Composed of p73, p15 and P57kip2 Is Associated with Poor Prognosis in Adult Patients with Philadelphia Chromosome Negative Acute Lymphocytic Leukemia (ALL).

Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. The high frequency of aberrant DNA methylation observed in patients with ALL, and the fact that these epigenetic changes are also present at relapse suggest that aberrant DNA methylation has a role in the pathogenesis of the disease. The identification of subgroups of patients with poor prognosis based on their methylation characteristics may translate in specific clinical interventions, such as the introduction of early allogeneic bone marrow transplantation or the use of hypomethylating agents. In initial studies, we have shown that methylation of more than 1 gene of a cell cycle regulatory pathway composed of p73, p15 and p57KIP2 occurs in close to 25% of patients, and is associated with a poor prognosis in patients with Philadelphia chromosome negative (Ph negative) disease (Blood 2003; 101:4131). Of importance, protein expression of this pathway was associated with a significant better prognosis by multivariate analysis (JCO 2005; 23:3932–9). To confirm the prognostic value of methylation of p73, p15 and p57KIP2, we are conducting an NCI-sponsored large-scale study of 300 patients wit Ph negative ALL at the time of initial presentation. As part of this study, and based on the original statistical design, we have performed an interim analysis of the first 93 patients studied. The objective of this interim analysis was to terminate this study early in case of a high likelihood that the study will be negative: that is that methylation of triad of genes is not associated with a poor prognosis. To detect DNA methylation, we have developed a new real-time bisulfite PCR assay. This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2 ug of DNA for each gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon gamma (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. The characteristics of the patients are similar to those of the initially reported cohort of patients (Blood 2003; 101:4131–6). The median overall survival (OS) of the whole group was 166 weeks. Out of the 93 patients, 88 (94%) achieved a complete remission. And out of these 88 patients, 37 (42%) eventually relapsed. The median disease-free survival was 127 weeks. P57KIP2 was methylated in 24% of patients, p15 in 34% and p73 in 21%. Methylation of more than 1 gene of this triad was observed in 19% of patients. This subgroup of patients had a median OS of 50 weeks. By multivariate analysis, methylation of more than 1 gene of this pathway (hazard ratio 4.07, 95% CI 1.63–7.5, p=0.003), presence of the CALLA antigen, older age, increased WBC and lower platelet count were associated with a significantly worse prognosis. Based on this interim analysis, this study continues, and will be completed by the time of the meeting. These results indicate that analysis of methylation markers may have a role in the prognostication of patients with Ph negative ALL.

Detection of Aberrant DNA Methylation in Acute Lymphocytic Leukemia (ALL) Using a Real-Time Polymerase Chain Reaction (PCR) Assay.

Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. DNA methylation of specific subsets of genes is associated with poor prognosis and is stable in a majority of patients (pts) at the time of relapse (Clinical Cancer Res 2002; 8:1897), and therefore tracking these epigenetic marks during remission may predict for relapse risk. Most commonly used methods to detect DNA methylation exploit the availability of sodium bisulfite that transforms unmethylated C into A/T leaving methylated C as such. This has allowed the development of several techniques that use conventional PCR or sequencing to detect methylated alleles. Although there are significant differences in the sensitivity and specificity of these assays, with bisulfite sequencing considered the gold standard, most of them are labor intensive and require several days to be performed. To circumvent some of these problems, we have developed a real-time bisulfite PCR assay to detect methylation of p57KIP2, p73, and p15 in samples obtained from bone marrows in pts with ALL at remission. Methylation of these genes has been shown to be common in ALL and to predict for poor prognosis at initial presentation (Blood 2003; 101:4131). This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2μg of DNA for each target gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon γ (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. Methylation density is defined as: Methylation (%) = 2CT of target gene / 2 CT of EFM x 100. CT is the number of cycles threshold, and EFM the estimated 100% fully methylation (EFM) of the target gene in control experiments. Using DNA artificially methylated with SssI in dilution experiments with unmethylated DNA, p57, p15 and p73 methylation density could be detected at dilutions of 0.2%, 1.2% and 0.1%, respectively. We then studied the methylation status of 30 cell lines (22 hematopoietic and 8 no-hematopoietic). Methylation of p57, p15 and p73 gene was detected in 17(57%), 19(63%) and 16(53%) cell lines respectively. Methylation of 3 genes, 2 genes and 1 gene was observed in 9(30%), 8(27%) and 7(23%) cell lines respectively. DNA methylation of p15 and p73 was not detected in the marrow from 6 healthy volunteers but p15 was detected (0.1% methylation) in 1 out of 6 of these specimens. Subsequently, we studied 50 pts with ALL at the time of remission. We found the frequencies of p57, p15 and p73 gene to be 2(4%), 26(52%) and 10(20%), respectively. There was a trend towards a better overall survival for pts with methylation of 0 or 1 gene (209 weeks) compared with those with methylation of 2 or more genes (71 weeks), p=0.1. In conclusion, the real-time bisulfite PCR described here allows for the rapid detection of aberrant DNA methylation in samples obtained at the time of remission in pts with ALL and may have a role in the development of techniques to assess minimal residual disease in this group of pts.

Resonant Multisensor System for High-Throughput Determinations of Solvent/Polymer Interactions

Solvent-resistant polymers are important in numerous research, engineering, and consumer applications. To address the limitations of existing methods of evaluation of polymer solubility and solvent resistance, we developed and built a 6 x 4 array of resonant acoustic-wave sensors operating in the thickness shear mode (TSM). The application of this system makes possible analysis of nanogram quantities of polymers in small amounts of solvent and permits the simultaneous analysis of multiple samples, such as those produced in combinatorial polymerization reactions. These parallel determinations of polymer/solvent interactions eliminate errors associated with serial determinations. During the periodic exposure of the TSM crystals to polymer/solvent combinations, the mass increase of the crystal is determined, which is proportional to the amount of polymer dissolved and deposited onto the sensor from a polymer solution. We demonstrate our sensor system for reliable quantification of solubility of several types of polymers in various solvents. The high mass sensitivity of our resonant TSM sensors (10 ng), use of only a minute volume of a solvent (< 2 mL), and parallel operation (matching a layout of available 24 well plates) make this system a good fit with available polymer combinatorial synthesis equipment.