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Method for detecting analytes by means of an analyte/polymeric activator bilayer arrangement

An analyte and activator technology, applied in the field of analytical sensors, can solve problems such as difficult detection, high background signal, and low sensitivity

Inactive Publication Date: 2006-12-06
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been shown that detection of larger nucleic acid molecules such as genomic DNA is difficult using these methods due to the high background signal, which results in rather low sensitivity at the pM or even nM level

Method used

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  • Method for detecting analytes by means of an analyte/polymeric activator bilayer arrangement
  • Method for detecting analytes by means of an analyte/polymeric activator bilayer arrangement
  • Method for detecting analytes by means of an analyte/polymeric activator bilayer arrangement

Examples

Experimental program
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Embodiment 1

[0116] Embodiment 1: the detection of nucleic acid

[0117]Generally, nucleic acid detection according to the present invention is performed as shown in FIG. 1 . First, a mixture of thiolated oligonucleotides (also carrying biotin modifications as labels) used as capture molecules (20) and thiol molecules used as blocking agents (15) to reduce background was immobilized on the surface of a gold electrode ( 10) on. The electrodes are then exposed to a solution supposedly containing the analyte of interest (30). After hybridization to its complementary biotinylated target DNA (ie, capture molecule), the enzyme conjugate is bound via an avidin-biotin interaction (50). Finally, the redox polymer (40) is brought onto the electrode surface by layer-layer electrostatic self-assembly. The redox polymer layer electrochemically activates the enzymatic label bound to the target DNA. The current generated by the catalytic oxidation of the substrate in the presence of the substrate mol...

Embodiment 11

[0118] Example 1.1: Extraction of mRNA from Rat Tissue and Synthesis of Biotinylated cDNA

[0119] Using Dynabeads® mRNA DIRECT TM Kit (Dynal ASA, Oslo, Norway), rat liver mRNA extraction was performed according to the manufacturer's instructions. For reverse transcription (RT), 10 ng of this mRNA was used in a total volume of 20 μl containing Sigma-Aldrich's 1×eAMV buffer (50 mM Tris-HCl, pH 8.3, 40 mM KCl, 8.0 mM MgCl 2 , 1 mM DTT), 500 μM of each dNTP, 1.0 μM antisense primer, 20 U RNase inhibitor and 20 U enhanced avian myeloblastosis virus reverse transcriptase (eAMV). The samples were incubated in a DNA thermal cycler (Gene Amp PCR System 9700, Applied Biosystems, Foster City, CA, USA) at 56°C for 50 min, and the obtained cDNA was directly used as a template for PCR amplification.

[0120] PCR was performed using 2.0 μl RT-reaction mix in a total volume of 50 μl containing Sigma-Aldrich's 1×AccuTaq buffer (5 mM Tris-HCl, 15 mM ammonium sulfate, pH 9.3, 2.5 mM MgCl 2 ...

Embodiment 12

[0124] Example 1.2: Capture probe immobilization and evaluation of monolayer quality

[0125] Prior to the detection of DNA analytes, a mixture of thiolated oligonucleotides and thiol molecules used as capture probes was immobilized on the gold electrode surface by self-assembly. To minimize absorption associated with non-hybridization of target DNA, anionic thiol molecules are used to form a mixed monolayer of blocking components. Use the following capture probes: for GAPDH detection, 5′-T 12 TTACTCCTTGGAGGCCATGTAGG-3' (SEQ ID NO: 5); and 5'-T 12 ATGGTGAAGGTCGGTGTCAACGG-3' (SEQ ID NO: 6); for TP53 detection, 5'-T 12 ATGGAGGATTCACAGTCGGA-3' (SEQ ID NO: 7) and 5'-T 12 TCAGTCTGAGTCAGGCCCCA-3' (SEQ ID NO: 8); and as a control, 5'-T 12 CCTCTCGCGAGTCAACAGAAACG-3' (SEQ ID NO: 9). Oligonucleotides were thiolated using 11-mercaptoundecanoic acid according to routine procedures and assembled onto gold electrodes by exposing the cleaned electrodes to a 50 μM oligonucleotide solutio...

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Abstract

The invention relates to the field of analytical sensors. In particular, the invention relates to a method for the detection of analytes in a sample by means of an electrode arrangement, which is characterized by the formation of a conductive bilayer of analytes and an agent for increasing the conductivity of said analytes on the surface of an electrode. The invention is also directed to an electrode arrangement useful for performing such method as well as to the use of such electrode arrangement as biosensor. Also disclosed is a novel class of redox polymers that are suitable for being used in the electrochemical detection of analytes. A method of making this class of polymers is also disclosed.

Description

[0001] The invention relates to the field of analytical sensors. In particular, the present invention relates to a method for detecting an analyte in a sample by means of an electrode arrangement characterized by the formation of a conductive double layer of the analyte and a reagent that increases the conductivity of the analyte on the electrode surface. The invention also relates to electrode arrangements for carrying out such methods, and to the use of such electrode arrangements as biosensors. Background of the invention [0002] Detection and quantification of analytes such as macromolecular biopolymers are fundamental methods in analytical chemistry as well as in biochemistry, food engineering or medicine. By far the most common methods used to determine the presence and concentration of biopolymers include detection by autoradiography, fluorescence, chemiluminescence or bioluminescence and electrochemical techniques (for example in Bakker, E. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/00C12Q1/18C12Q1/26C12Q1/54G01N33/543G01N27/26
CPCG01N33/5438C12Q1/004G01N33/54393C12Q1/6825B82Y15/00C12Q1/26B82Y30/00C12Q2565/519C12Q2563/113
Inventor 高志强谢虹詹宗原余袁宏
Owner AGENCY FOR SCI TECH & RES
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