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1.
Biosens Bioelectron ; 35(1): 87-93, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22405908

ABSTRACT

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target-probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target-probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.


Subject(s)
Biosensing Techniques/methods , DNA, Bacterial/analysis , Microbiological Techniques/methods , Bacteriological Techniques/instrumentation , Bacteriological Techniques/methods , Bacteriological Techniques/statistics & numerical data , Base Sequence , Biosensing Techniques/instrumentation , Biosensing Techniques/statistics & numerical data , Computer Systems , DNA, Bacterial/genetics , Data Interpretation, Statistical , Electric Impedance , Equipment Reuse , Escherichia coli/genetics , Escherichia coli/isolation & purification , Genes, Bacterial , Microbiological Techniques/instrumentation , Microbiological Techniques/statistics & numerical data , Polymerase Chain Reaction
2.
Article in English | MEDLINE | ID: mdl-22254908

ABSTRACT

In previously published work [1] we presented a real-time electrochemical impedance biosensor prototype system and a state-space estimation algorithm for signal quantification. Experiments in the interim have revealed some algorithm failure modes which reduced the reliability and repeatability of quantification. The present work describes a related algorithm that introduces constraints based on a priori knowledge of the expected signals predicted by the biosensor signal model. The improvements in reliability and repeatability bring the system close to deployment for real-world trials.


Subject(s)
Algorithms , Biosensing Techniques , Reproducibility of Results , Signal Processing, Computer-Assisted
3.
Article in English | MEDLINE | ID: mdl-21096210

ABSTRACT

We describe our real-time, label-free, electrochemical impedance biosensor system with an emphasis on the use of an impedance response signal model to quantify assays. The signal processing for estimating model parameters from noisy data and the quantitative verification against target concentration and affinity are also presented.


Subject(s)
Biosensing Techniques , Electric Impedance , Algorithms , Computers , DNA, Single-Stranded/genetics , Electrochemistry/methods , Electrodes , Equipment Design , Escherichia coli/genetics , Kinetics , Nonlinear Dynamics , Signal Processing, Computer-Assisted , Time Factors
4.
Biosens Bioelectron ; 22(9-10): 1853-60, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-16891109

ABSTRACT

Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes. First, microarray probes are hybridized to biotin-labeled targets, second, the HRP-streptavidin conjugate binds to biotin, and enzymatic oxidation of the electron donor substrate then occurs. The detection current is generated due to electro-reduction of the HRP reaction product, and it is measured with the CombiMatrix ElectraSense Reader. Performance of the ElectraSense platform has been characterized using gene expression and genotyping assays to analyze: (i) signal to concentration dependence, (ii) assay resolution, (iii) coefficients of variation, (CV) and (iv) array-to-array reproducibility and data correlation. The ElectraSense platform was also compared to the standard fluorescent detection, and good consistency was observed between these two different detection techniques. A lower detection limit of 0.75 pM was obtained for ElectraSense as compared to the detection limit of 1.5 pM obtained for fluorescent detection. Thus, the ElectraSense platform has been used to develop nucleic acid assays for highly accurate genotyping of a variety of pathogens including bio-threat agents (such as Bacillus anthracis, Yersinia pestis, and other microorganisms including Escherichia coli, Bacillus subtilis, etc.) and common pathogens of the respiratory tract (e.g. influenza A virus).


Subject(s)
Electrochemistry , Gene Expression Profiling , Oligonucleotide Array Sequence Analysis , Bacteriophage lambda/genetics , Electrochemistry/instrumentation , Gene Expression Profiling/instrumentation , Genotype , Humans , Oligonucleotide Array Sequence Analysis/instrumentation
5.
Lab Chip ; 6(8): 1052-5, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16874377

ABSTRACT

We show that multiple enzyme tags may be used in an immunoassay format or for the detection of sequence-specific DNA on microarrays. The assays may be multiplexed and monitored under separate solution and voltage differences. Thus, the detection method relies on an electrochemical detection format, whereby multiple enzymes can be sensed. In our case we utilize horseradish peroxidase, laccase, and glucose dehydrogenase as enzymes attached to specific antibodies or to streptavidin.


Subject(s)
Antibodies/chemistry , Oligonucleotide Array Sequence Analysis , Oxidoreductases/chemistry , Streptavidin/chemistry , Electrochemistry
6.
Anal Chem ; 78(6): 1980-6, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16536436

ABSTRACT

A DNA microarray with 12,000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated into the cartridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps in genomic analysis.


Subject(s)
Bacteriophage lambda/genetics , Gene Expression Profiling/instrumentation , Microfluidics/methods , Oligonucleotide Array Sequence Analysis/methods , Escherichia coli/genetics , Microfluidics/instrumentation , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis/instrumentation , Sensitivity and Specificity
7.
J Biochem Biophys Methods ; 59(2): 181-7, 2004 May 31.
Article in English | MEDLINE | ID: mdl-15163529

ABSTRACT

A CMOS fabricated silicon microchip was used as a platform for immunoassays and DNA synthesis and hybridization. The chip is covered with a biofriendly matrix wherein the chemistries occur. The active silicon chip has over 1000 active electrodes that can be individually addressed for both synthesis of DNA and protein attachment to a membrane on the chip surface. Additionally, the active chip can be further used for the detection of various analytes at the chip surface via digital read out resulting from the redox enzymes on the captured oligonucleotide or antibody.


Subject(s)
DNA/analysis , DNA/genetics , Horseradish Peroxidase/metabolism , Immunoassay/methods , Oligonucleotide Array Sequence Analysis/methods , Bacteriophages/immunology , Bacteriophages/isolation & purification , Electrochemistry , Electrodes , Humans , Immunoassay/instrumentation , Light , Oligonucleotide Array Sequence Analysis/instrumentation , Orosomucoid/analysis , Orosomucoid/immunology , Solutions
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