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Methods of producing homogeneous plastic-adherent aptamer-magnetic bead-fluorophore and other sandwich assays

A sandwich analysis, fluorophore technology, applied in biochemical equipment and methods, microbial determination/inspection, biological testing, etc., can solve problems such as not very good effect, not very good adhesion effect, etc.

Inactive Publication Date: 2011-08-24
OTC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, immunomagnetic ("antibody-magnetic bead") sandwich analytes do not adhere well to polystyrene at neutral or acidic pH, possibly because protein antibodies bind plastic or polystyrene at neutral or acidic pH. The reason why the adhesion effect of the glass material is not very good

Method used

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  • Methods of producing homogeneous plastic-adherent aptamer-magnetic bead-fluorophore and other sandwich assays
  • Methods of producing homogeneous plastic-adherent aptamer-magnetic bead-fluorophore and other sandwich assays
  • Methods of producing homogeneous plastic-adherent aptamer-magnetic bead-fluorophore and other sandwich assays

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] One-step wash-free ultrasensitive detection of Campylobacter jejuni (C. jejuni) in various food matrices

[0056] The present invention is used to detect such as Figure 2-4 Pure buffers and various food matrices shown contained as few as 2 live or dead cells of C. jejuni (a common foodborne pathogen). Among these analytes, there were two distinct C. jejuni sequences (represented by C2 and C3 or SEQ ID NO.2 and SEQ ID NO.3) whose 2'-amino groups were modified during solid-phase DNA synthesis , and then combined with 1,000 tosyl-M280 (diameter 2.8 μm) Dynal (Invitrogen) magnetic beads or 0.24 pL Q-dot 655ITK reagent (Invitrogen) for each test. The C2 aptamer (SEQ ID 2) is used for capture on the surface of tosyl-magnetic beads, and the C3 aptamer (SEQ ID NO.3) is passed through BS 3 (bis-suberate bifunctional linker from Pierce Chemical Co.) was used as a reporter reagent after conjugation with Q-dot 655ITK reagent. These reagents are purified, mixed, and lyophilized ...

Embodiment 2

[0058] One-step wash-free ultrasensitive detection of Escherichia coli O157:H7 and other toxin-producing (shiga or vero toxin) E. coli in various food matrices

[0059] By combining the aptamer with the outer sugar of O157 lipopolysaccharide (LPS) and the H7 flagellar antigen, the invention can be used to detect enterohemorrhagic E. coli O157:H7 in various foods or on the surface of foods. Aptamer sequences from SEQ ID NOs.7-20 can be used for capture (aptamer-magnetic bead conjugate) or reporter (aptamer-fluorophore conjugate) function, and for detecting E. coli O157:H7. Alternatively, outer membrane proteins (OMPs) shared by many E.coli can also be used for aptamer-magnetic bead capture (or identification) of E.coli bacterial cells, followed by LPS-specific aptamer-quantum dot The reporter reagent is used for specific identification of E.coli strains or serotypes. The aptamer SEQ ID NOs.279-322 can be used for the recognition and capture of E.coli OMP. In another example,...

Embodiment 3

[0061] One-step no-wash ultrasensitive detection of Listeria monocytogenes (Listeria monocytogens) in various food matrices

[0062] By combining with listerolysin (listerolysin, LO) surface protein, the present invention can be used to detect deadly bacteria L. monocytogens in various foods or on the surface of foods. Aptamer sequences from SEQ ID NOs.21-52 can be selected for capture (aptamer-magnetic bead conjugate) or reporter (aptamer-fluorophore conjugate) function, and for detection of LO and L. monocytogens.

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Abstract

Methods are described for assembly of DNA aptamer-magnetic bead ('MB') conjugate plus aptamer-quantum dot ('QD') aptamer-fluorescent nanoparticle or other aptamer-fluorophore, aptamer-chemiluminescent reporter, aptamer-radioisotope or other aptamer-reporter conjugate sandwich assays that enable adherence to glass, polystyrene and other plastics. Adherence to glass or plastics enables detection of surface-concentrated partitioning of fluorescence versus background (bulk solution) fluorescence in one step (without a wash step) even when the external magnetic field for concentrating the assay is removed. This assay format enables rapid, one-step (homogeneous) assays for a variety of analytes without wash steps that do not sacrifice sensitivity.

Description

[0001] Cross References to Related Applications [0002] This application is based on, and claims priority from, US Provisional Application Nos. 61 / 066,506 and 61 / 132,147, the entire contents of which are incorporated herein by reference. technical field [0003] The present invention relates to the related fields of aptamer-based and nucleic acid-based diagnostic reagents. More specifically, the present invention relates to the preparation of self-assembling DNA aptamer-magnetic bead ("MB") conjugates or other aptamer-fluorophore conjugate sandwich analytes conjugated to aptamer-quantum dots ("QD") and methods of use wherein the analyte naturally adheres to glass and certain plastics such as polystyrene (or its derivatives) for one-step (homogeneous) detection without washing, even after removal of external magnetic beads No washing required. The binding between aptamers and magnetic beads or quantum dots and other fluorophores can be achieved through simple chemical bindin...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q1/6825C12Q1/6804C12Q2563/149C12Q2563/143C12Q2525/205C12Q2563/107C12N15/115G01N33/52C12N2310/16
Inventor 约翰·G·布鲁诺
Owner OTC BIOTECH
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