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Continuous cell detection by isotachophoresis

a cell detection and isotachophoresis technology, applied in the field of isotachophoresis system, can solve the problems of high labor intensity, high labor intensity, and time-consuming conventional bacteria detection methods,

Active Publication Date: 2017-06-29
TECHNION RES & DEV FOUND LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent is about a system and method that allows for the detection and sampling of cells based on their membranal components. The system includes a protein that binds to a membranal component, a flow channel for carrying a liquid sample containing the cells, and an isotachophoresis apparatus for separating the cells on the basis of their membrane components. The method involves using a labeled protein to focus the cells onto the apparatus, applying a counter flow to keep the protein in a stationary position, and introducing the sample containing the cells of interest. By detecting or sampling the cells, this technology provides a safe and efficient way to analyze and manipulate cells of interest.

Problems solved by technology

The system quickly establishes a strong gradient in electric field at the ITP interface, due to the non-uniform conductivity profile.
The conventional bacteria detection methods—e.g. sample cultivation, genotypic detection methods and immunoassays—are time consuming, comprise of several manual steps, and require highly trained personnel.
However, despite the many advantages of this technology, to date, most microfluidic assays cannot perform continuous analysis and are limited by their ability to analyze only a single and finite amount of sample, and are thus coupled to significant sample preparation, e.g. by filtration or centrifugation.

Method used

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  • Continuous cell detection by isotachophoresis
  • Continuous cell detection by isotachophoresis
  • Continuous cell detection by isotachophoresis

Examples

Experimental program
Comparison scheme
Effect test

example 1

Microfluidic Assay for Continuous Real-Time Pathogen Detection Using Antimicrobial Peptides and Isotachophoresis

[0101]The basic assumption for the following experiment was that PNAs targeting bacteria by non-specific binding to their negatively charged outer-membrane can be used as the protein as described herein and the bacteria to be detected can be the cell to be detected as described herein.

[0102]An ITP system such as illustrated in FIG. 1 was used for the current experiments. The system included the settings as described herein. Specifically, positively charged labeled AMPs are universal probes for labeling and detecting bacteria. Using cationic ITP focusing, a high concentration zone of AMPs within a microchannel was formed. A pressure driven flow countering electromigration was applied to hold the zone stationary. Through this “virtual reaction chamber” the sample of interest was flowing (from the LE reservoir). Any bacteria present in the sample was simultaneously labeled by...

example 2

Labeling Efficiency

[0114]The sensitivity of the assay is directly affected by the flow rate, as well as the labeling efficiency of bacteria as it passes through the AMPs confined at the LE-TE interface. To characterize the latter, the assay was performed on tap water samples spiked with bacteria which were prelabeled with SYTO9. After stabilizing the assay, the counterflow and voltage were simultaneously stopped, such that all bacteria remain stationary. The channel was imaged at 10 stations downstream of the ITP interface zone. At each station, images were taken using two filters: 480 / 535 for detecting the bacteria prelabeled with SYTO9 and 545 / 605 for detecting the bacteria which were labeled on-chip by AMPs. SYTO9 emits at a wavelength of 500 nm, which is sufficiently shifted from the 579 nm emission of the TAMRA labeled AMPs, and thus, it is possible to measure the number of bacteria which were successfully labeled and compare it with the total number of bacteria that passed thr...

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Abstract

The present invention provides a system including: a protein having a domain that binds a membranal component; an inlet for sample flow, an Isotachophoresis (ITP) system and a flow generating means connected or coupled to the aqueous parts of the ITP. The invention also provides a method for detecting and or sorting cells with this system.

Description

FIELD OF INVENTION[0001]This invention is directed to; inter alia, an isotachophoresis system for the detection and / or separation of cells based on a membranal component detectable by a protein.BACKGROUND OF THE INVENTION[0002]Isotachophoresis[0003]Isotachophoresis (“ITP”) is a variant of electrophoresis, characterized by the fact that separation is carried out in a discontinuous buffer system. Sample material to be separated is inserted between a “leading electrolyte” and a “terminating electrolyte” or mixed in any of these, the characteristic of these two buffers being that the leader has to have ions of net mobility higher than those of sample ions, while the terminator must have ions of net mobilities lower than those of sample ions. In such a system, sample components sort themselves according to decreasing mobilities from leader to terminator, in a complex pattern governed by the so-called Kohlrausch regulating function. The process has been described repeatedly, as for instan...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N27/447
CPCG01N27/44769G01N27/44765G01N27/44743G01N27/44791B01L3/50273B01L2200/0652B01L2300/0645B01L2300/0816B01L2300/0867B01L2400/0421B01L2400/0487G01N27/44726G01N27/44756G01N33/561G01N33/56911B01L2300/06B01L2300/0627B01L2300/0887B01L2300/12B01L2400/043G01N27/44721
Inventor BERCOVICI, MORANSCHWARTZ, ORTAL
Owner TECHNION RES & DEV FOUND LTD