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Process to detect binding events on an electrode microarray using enzymes

A microarray and enzyme detection technology, applied in biochemical equipment and methods, measuring devices, biological testing, etc., can solve the problem of difficult to distinguish gray shadow signal true positive or false positive, fluorescence signal quenching, increased variability, etc. question

Active Publication Date: 2009-04-01
CUSTOMARRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Also, it can be difficult to tell whether a shade of gray or a barely perceptible signal is a true positive or a false positive
Finally, the fluorescent signal may be quenched, or the signal may be autoabsorbed by other labels in the vicinity of the bound target
Additional complexities associated with adopting photonic-based readers add to variability

Method used

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  • Process to detect binding events on an electrode microarray using enzymes
  • Process to detect binding events on an electrode microarray using enzymes
  • Process to detect binding events on an electrode microarray using enzymes

Examples

Experimental program
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Embodiment 1

[0228] Horseradish peroxidase

[0229] In the first immunological experiment, quinone, the product of HRP-catalyzed reaction, was detected by amperometric method. The experiment was carried out at -0.3 volts and platinum wire in an aqueous solution containing 0.05 moles of citric acid-sodium phosphate buffer, pH 5.0, and 0.2 moles of sodium sulfate. The concentration of catechol is 1 millimolar and the concentration of hydrogen peroxide is 1 millimolar. In order to connect HRP, probe DNA was synthesized in situ on the electrode microarray in a checkerboard pattern. Standard phosphoramidite chemistry and electrochemically generated acid used to remove protecting groups are used. The target DNA complementary to the probe DNA is labeled with biotin. Incubate with 1 nanomole of target DNA in 2XPBST solution at 40 degrees Celsius for 1 hour to hybridize the target DNA with the probe DNA.

[0230] Add HRP with a streptavidin tag to link biotin with HRP-streptavidin complex solution. The...

Embodiment 2

[0232] Laccase

[0233] In the second immunoassay, quinone, the product of the reaction catalyzed by laccase, was detected by amperometric method. The experiment was carried out at -0.3 volts with platinum wire in an aqueous solution containing 0.05 moles of citric acid-sodium phosphate buffer, pH 5.0, and 0.2 moles of sodium sulfate. The concentration of catechol is 1 millimolar, and the oxygen concentration has not been determined, but it is determined by the amount naturally present in the solution. In order to connect the laccase, target DNA was synthesized in situ on the electrode microarray in a checkerboard pattern. Standard phosphoramidite chemistry and electrochemically generated acid used to remove protecting groups are used. The target DNA complementary to the probe DNA is labeled with biotin. Incubate with 1 nanomole of target DNA in 2XPBST solution at 40 degrees Celsius for 1 hour to hybridize the target DNA with the probe DNA. Incubate at 25 degrees Celsius for 60 mi...

Embodiment 3

[0236] β-galactosidase immunoassay

[0237]In the immunoassay performed on the electrode microarray, β-galactosidase was used as the enzyme and the substrate was X-Gal. Using biotinylated β-galactosidase. The reaction was carried out in phosphate buffered saline at pH 7.0. The buffer concentration is 0.01M. To prepare the solution, the selected amount of X-Gal was dissolved in DMF because it is very soluble in DMF, but has low solubility in water. DMF containing X-Gal was added to the aqueous buffer. The final concentration of X-Gal is about 0.1 millimolar, which is close to the saturation concentration. During the process of adding DMF containing X-Gal to the buffer, stir the buffer vigorously to prevent X-Gal precipitation due to the limited water solubility of X-Gal. If the DMF solution of X-Gal is added without stirring vigorously, X-Gal will precipitate. The instability of this solution requires the preparation of a fresh solution every day. A ferric cyanide / ferrous solution ...

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Abstract

The present invention provides a process to detect binding events on an electrode microarray. A microarray is provided having addressable electrodes and two or more different types of capture complexes at sites corresponding to the electrodes. The capture complexes capture analytes. Enzymes are attached to form a reporter complex. Substrate solutions are sequentially contacted to make enzyme products that are detectable at the electrodes by a difference in the electrical response at electrodes having the enzyme product and those not having the enzyme product. The enzyme product may be a solid deposition product. The electrical properties of electrodes on the microarray are read for the presence of the enzyme product by sequentially switching each electrode held at a constant voltage to ground and then back to the constant voltage.

Description

[0001] Statement on Federally Funded Research [0002] Part of this invention was funded by the Department of Defense Phase II SBIR grant number DAAD13-00-C-0033 and the Department of Defense contract number W911SR-04-C-0022. Technical field [0003] The present invention provides a method for electronically detecting the binding event between a probe attached at a known position in a microarray device with electrodes and a target in a solution. The enzyme part and a substrate solution that can change the electrical characteristics of the electrode containing the enzyme part are used for detection. Background of the invention [0004] In the field of biotechnology research and development, microarrays have become an important analytical research tool. Generally, a microarray is an array of miniaturized locations on a solid surface that is generally flat. As part of the preparation of the microarray, pre-synthesized molecules, including biomolecules, may be attached to the location...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/00G01N33/48G01N33/50
CPCG01N33/5438C12Q1/001C12Q1/6825C12Q1/6837C12Q2565/607C12Q2563/125C12Q2537/149
Inventor K·迪尔J·J·库珀A·吉尼蒂利斯K·M·罗斯
Owner CUSTOMARRAY INC