Dendrimer Conjugates for Determining Membrane Retention Level and/or Pore Structure

Patent metadata

Field	Value
Publication number	US-2017009290-A1
Application number	US-201514796150-A
Country	US
Kind code	A1
Filing date	Jul 10, 2015
Priority date	Jul 10, 2015
Publication date	Jan 12, 2017
Grant date	—

Abstract

Dendrimer conjugates for determining membrane retention level and/or pore structure, methods of determining membrane level/pore structure, and kits including dendrimer conjugates are disclosed.

First claim

1 . A method for determining retention level and/or pore structure of a membrane, the method comprising:
(a) passing a challenge solution comprising single-stranded DNA conjugated to dendrimers (dendrimer conjugates), through a microporous membrane, to provide a permeate solution comprising the dendrimer conjugates; (b) obtaining equal volums of:
i. the challenge solution comprising dendrimer conjugates; and,
ii. the permeate solution comprising dendrimer conjugates;
(c) combining (i) with PCR reagents to provide a challenge solution/PCR mix, and combining (ii) with PCR reagents to provide a permeate solution/PCR mix; (d) performing a plurality of PCR cycles using each of the challenge solution/PCR mix, and the permeate solution/PCR mix, to amplify the single-stranded DNA; (e) measuring single-stranded DNA concentration, which correlates to dendrimer conjugate concentration; (f) quantifying the dendrimer conjugate concentrate in the challenge solution (quantCo); and quantifying the dendrimer conjugate concentration in the permeate solution (quantCp); and, (g) determining the retention level and/or pore structure of the membrane based on a comparison of the quantCp to the quantCo.

2 . The method of claim 1 , wherein the PCR cycles are qPCR cycles.

3 . The method of claim 1 , wherein (g) comprises comparing log reduction values for quantCp and quantCo.

4 . The method of claim 1 , wherein the dendrimer conjugates have a 1:1 ratio of single-stranded DNA to dendrimer.

5 . The method of claim 1 , comprising passing the challenge solution through the microporous membrane at a constant flow rate.

6 . The method of claim 1 , comprising passing the challenge solution through the microporous membrane at a constant pressure.

7 . The method of claim 1 , wherein (c) and (d) comprise using a device comprising a rotatable circular cartridge having a plurality of reaction chambers, and a reservoir, the reaction chambers being connected to the reservoir via channels, wherein at least a portion of the reaction chambers comprise primers; and a heating plate having at least two distinct zones that can be heated to at least two different temperatures, the method comprising:
at least partially filling the reservoir with a fluid containing dendrimer conjugates and PCR reagents for carrying out an amplification reaction, with the exception of primers; distributing the fluid to the reaction chambers comprising the primers therein; and, rotating the rotatable circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of the heating plate, wherein the single-stranded DNA is amplified.

8 . The method of claim 7 , wherein the heating plate has at least three distinct zones that can be heated to at least three different temperatures, and the method includes rotating the rotatable circular cartridge to successively bring the contents of each reaction chamber to the at least three temperatures defined by the at least three distinct zones of the heating plate.

9 . The method of claim 7 , wherein at least a portion of the reaction chambers comprise probes in addition to the primers.

10 . A kit for determining retention level and/or pore structure of a membrane comprising:
a solution comprising a plurality of dendrimers conjugated to single-stranded DNA; a rotatable circular cartridge having a plurality of reaction chambers containing dried primers, and a reservoir, wherein the reaction chambers are connected to the reservoir via channels; and a qPCR master mixture.

11 . The kit of claim 10 , wherein the reaction chambers container the dried primers and dried probes.

12 . The method of claim 2 , wherein (g) comprises comparing log reduction values for quantCp and quantCo.

13 . The method of claim 2 , wherein the dendrimer conjugates have a 1:1 ratio of single-stranded DNA to dendrimer.

14 . The method of claim 3 , wherein the dendrimer conjugates have a 1:1 ratio of single-stranded DNA to dendrimer.

15 . The method of claim 2 , comprising passing the challenge solution through the microporous membrane at a constant flow rate.

16 . The method of claim 2 , comprising passing the challenge solution through the microporous membrane at a constant pressure.

17 . The method of claim 3 , comprising passing the challenge solution through the microporous membrane at a constant flow rate.

18 . The method of claim 3 , comprising passing the challenge solution through the microporous membrane at a constant pressure.

19 . The method of claim 2 , wherein (c) and (d) comprise using a device comprising a rotatable circular cartridge having a plurality of reaction chambers, and a reservoir, the reaction chambers being connected to the reservoir via channels, wherein at least a portion of the reaction chambers comprise primers; and a heating plate having at least two distinct zones that can be heated to at least two different temperatures, the method comprising:
at least partially filling the reservoir with a fluid containing dendrimer conjugates and PCR reagents for carrying out an amplification reaction, with the exception of primers; distributing the fluid to the reaction chambers comprising the primers therein; and, rotating the rotatable circular cartridge to successively bring the contents of each reaction chamber to the at least two temperatures defined by the at least two distinct zones of the heating plate, wherein the single-stranded DNA is amplified.

20 . The method of claim 8 , wherein at least a portion of the reaction chambers comprise probes in addition to the primers.

Assignees

• Pall Corp

Inventors

• Ahmad Farhan
• Qiu Jian M
• Singh Amarnauth
• Mish Barbara

Classifications

• G01N2015/086

  of films, membranes or pellicules · CPC title

• G01N15/08

  Investigating permeability, pore-volume, or surface area of porous materials · CPC title

• C12Q1/68 Primary

  involving nucleic acids · CPC title

• B01D65/102

  Detection of leaks in membranes · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title