Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays

Goff, Gaelle C. Le; Desmet, Cloé; Brès, J.; Rigal, Dominique; Blum, Loı̈c J.; Marquette, Christophe A.

doi:10.1016/j.bios.2010.06.057

Cited by 6 publications

(7 citation statements)

References 19 publications

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“…The quantitative target detection was demonstrated (Figure 6b) with a limit of detection of 50 pmol L −1 (calculated as three times standard deviation of the blank). This compares well with the sensitivity previously demonstrated in our group for microarrays spotted on a porous membrane, highly efficient for probe immobilization, using the same probe/target couple 26. Moreover, the specificity of target capture was demonstrated by dispensing a sample containing a single target sequence on a matrix of hydrogel dots containing two different probe sequences (Figure 6c).…”

Section: Resultssupporting

confidence: 88%

Shrinking Hydrogel‐DNA Spots Generates 3D Microdots Arrays

Goff

Blum

Marquette

2013

Macromolecular Bioscience

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This report describes a straightforward approach for the achievement of sub-100 micrometers size hydrogel dots supporting DNA immobilization. Hydrogel-DNA spots are arrayed and UV-crosslinked on PolyShrink, an innovative polymer material having the remarkable property of isotropically shrinking under high temperature. Curing the microarray enables then spot miniaturization, resulting in 6 µm thick and 60 µm wide hydrogel dots in which oligonucleotides are immobilized in a 3D hydrophilic environment. The probe immobilization within the hydrogel network and its capacity to detect targets specifically and quantitatively is demonstrated using chemiluminescent as well as colorimetric detection techniques. The hydrogel material improves probe accessibility within the spot, leading to an enhanced sensitivity.

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Section: Resultssupporting

confidence: 88%

Shrinking Hydrogel‐DNA Spots Generates 3D Microdots Arrays

Goff

Blum

Marquette

2013

Macromolecular Bioscience

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“…Agitation of aqueous mixtures of analyte by shaking or manual aspiration/dispensing only marginally enhanced detection of live bacteria but had no impact on the detection of Stx1 indicating that intact cells, and not fragments or relatively small biomolecules, were solely influenced by the applied centrifugal force. A prospective alternative to centrifugation may be employing multiwell plates that incorporate biorecognition element arrayed filter membranes, a combination recently exhibited by [ 33 ]. Any process that forces analyte and antibody probe and/or subsequent reporter probe into close association will be advantageous to detection as exhibited by the method herein.…”

Section: Discussionmentioning

confidence: 99%

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates

Gehring¹,

Brewster²,

He³

et al. 2015

Sensors

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Antibody microarray is a powerful analytical technique because of its inherent ability to simultaneously discriminate and measure numerous analytes, therefore making the technique conducive to both the multiplexed detection and identification of bacterial analytes (i.e., whole cells, as well as associated metabolites and/or toxins). We developed a sandwich fluorescent immunoassay combined with a high-throughput, multiwell plate microarray detection format. Inexpensive polystyrene plates were employed containing passively adsorbed, array-printed capture antibodies. During sample reaction, centrifugation was the only strategy found to significantly improve capture, and hence detection, of bacteria (pathogenic Escherichia coli O157:H7) to planar capture surfaces containing printed antibodies. Whereas several other sample incubation techniques (e.g., static vs. agitation) had minimal effect. Immobilized bacteria were labeled with a red-orange-fluorescent dye (Alexa Fluor 555) conjugated antibody to allow for quantitative detection of the captured bacteria with a laser scanner. Shiga toxin 1 (Stx1) could be simultaneously detected along with the cells, but none of the agitation techniques employed during incubation improved detection of the relatively small biomolecule. Under optimal conditions, the assay had demonstrated limits of detection of ~5.8 × 105 cells/mL and 110 ng/mL for E. coli O157:H7 and Stx1, respectively, in a ~75 min total assay time.

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“…Hi-Fi plate spotting and hybridization procedures were described elsewere . Here, a two-step protocol (detailed in Supporting Information) was used for hybridization and labeling.…”

Section: Methodsmentioning

confidence: 99%

“…Our group previously reported a cost-efficient high-throughput analysis platform (HiFi-assay) for multiparametric nucleic acids assays, on the basis of a colorimetric detection of hybridization. , This tool, initially developed for high-throughput blood group genotyping assays, was designed in order to fulfill end-user needs (i.e., blood qualification centers): (i) the device is fully compatible with robotic platforms and thus answers throughput constrains (thousands of samples tested per day), and (ii) it is based on an enzymatic colorimetric detection process using alkaline phosphatase as label which reduces the cost per test. Although we obtained very satisfying results, detection can still be further improved.…”

mentioning

confidence: 99%

Enhanced Colorimetric Detection on Porous Microarrays Using in Situ Substrate Production

2011

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A new technique is reported for the enhanced colorimetric detection of multiplexed hybridization onto porous membrane-based microarrays. This approach combines the use of horseradish peroxidase (HRP) as a label together with a chromogen substrate and a local production of the hydrogen peroxide required for substrate oxidation. This in situ production of coreagent is obtained using glucose oxidase (GOx) directly immobilized within the microarray porous membrane mesh. The oxidation of glucose by the immobilized GOx produces hydrogen peroxide which itself enables the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) by HRP and yields a blue precipitate on positive spots. Thanks to a coreagent overconcentration within the membrane, this design drastically surpasses the performances of the standard TMB/H(2)O(2) kit used for peroxidase label detection. The obtained target limit of detection is then 50 times lower (20 pM) than the one obtained with the standard kit approach, and the dynamic range expands at least one decade. Furthermore, the developed method was shown to compete well with the widely used alkaline phosphatase-BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitro blue tetrazolium chloride) readout while minimizing background signal. The method was finally successfully applied to the multiplex detection of single nucleotide polymorphisms (SNPs) in complex PCR samples. The background lowering was impacted here positively on the SNPs' detection by increasing the complementary/noncomplementary signal ratio.

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Multipurpose high-throughput filtering microarrays (HiFi) for DNA and protein assays

Cited by 6 publications

References 19 publications

Shrinking Hydrogel‐DNA Spots Generates 3D Microdots Arrays

Shrinking Hydrogel‐DNA Spots Generates 3D Microdots Arrays

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates

Enhanced Colorimetric Detection on Porous Microarrays Using in Situ Substrate Production

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