Visible Detection of Microorganisms

Abstract

Methods of detecting very low levels of targets, such as cells, are provided. In some embodiments, for example, the methods can detect bacteria present in a sample at concentrations less than 25 cells/mL. The method involves detecting nanoparticle aggregation in the absence of the target.

1 . A method of determining whether a sample comprises a target, comprising:
 a) contacting the sample with a linker, wherein the linker comprises a first functionality and a plurality of second functionalities, wherein the first functionality is capable of binding to the target, and wherein each of the plurality of second functionalities is capable of binding to a third functionality;   b) contacting the sample from (a) with a plurality of nanoparticles, wherein each of the plurality of nanoparticles comprises a third functionality that is capable of binding to the second functionality, and wherein the linker is added in step (a) at a concentration less than the concentration of nanoparticles and sufficient to aggregate the nanoparticles added in step (b) in the absence of target; and   c) detecting nanoparticle aggregation in the sample from (b), wherein the absence of nanoparticle aggregation indicates that the sample comprises the target.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the linker is an antibody and the first functionality is the antigen binding region of the antibody. 
     
     
         6 . The method of  claim 5 , wherein the second functionality is biotin and the third functionality is streptavidin. 
     
     
         7 . The method of  claim 1 , wherein the nanoparticles are selected from gold nanoparticles, silver nanoparticles, platinum nanoparticles, magnetite nanoparticles, gold/iron alloy nanoparticles, and latex nanoparticles. 
     
     
         8 . The method of  claim 1 , wherein the target is selected from prokaryotic cells, eukaryotic cells, and parasites. 
     
     
         9 . The method of  claim 1 , wherein detecting nanoparticle aggregation comprises determining at least one characteristic selected from sample color, UV-VIS spectrum, UV-VIS peak wavelength, and absorbance. 
     
     
         10 . The method of  claim 9 , wherein the at least one characteristic of the sample from (b) is compared to at least one characteristic of a standard. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . A method of determining whether a sample comprises a target, comprising:
 a) contacting the sample with a linker, wherein the linker comprises a first functionality and a plurality of second functionalities, wherein the first functionality is capable of binding to the target, and wherein each of the plurality of second functionalities is capable of binding to a third functionality;   b) contacting the sample from (a) with a plurality of nanoparticles, wherein each of the plurality of nanoparticles comprises a third functionality that is capable of binding to the second functionality, and wherein the linker is added in step (a) at a concentration just in excess relative to the concentration of the nanoparticles added in step (b) and the nanoparticles do not aggregate in the absence of target; and   c) detecting nanoparticle aggregation in the sample from (b), wherein the presence of nanoparticle aggregation indicates that the sample comprises the target.   
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . The method of  claim 13 , wherein the linker is an antibody and the first functionality is the antigen binding region of the antibody. 
     
     
         18 . The method of  claim 17 , wherein the second functionality is biotin and the third functionality is streptavidin. 
     
     
         19 . The method of  claim 13 , wherein the nanoparticles are selected from gold nanoparticles, silver nanoparticles, platinum nanoparticles, magnetite nanoparticles, gold/iron alloy nanoparticles, and latex nanoparticles. 
     
     
         20 . The method of  claim 13 , wherein the target is selected from prokaryotic cells, eukaryotic cells, and parasites. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 13 , wherein detecting nanoparticle aggregation comprises determining at least one characteristic selected from sample color, UV-VIS spectrum, UV-VIS peak wavelength, and absorbance. 
     
     
         23 . The method of  claim 22 , wherein the at least one characteristic of the sample from (b) is compared to at least one characteristic of a standard. 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . A kit for determining whether a sample comprises a target, comprising (i) a linker, wherein the linker comprises a first functionality and a plurality of second functionalities, wherein the first functionality is capable of binding to the target, and wherein each of the plurality of second functionalities is capable of binding to a nanoparticle; (ii) a plurality of nanoparticles, wherein each of the plurality of nanoparticles comprises a third functionality that is capable of binding to the second functionality; and (iii) a standard, wherein the standard is a representation of at least one characteristic of a control reaction that comprises the linker and the plurality of nanoparticles, but not the target. 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . The kit of  claim 26 , wherein the linker is an antibody and the first functionality is the antigen binding region of the antibody. 
     
     
         31 . The kit of  claim 30 , wherein the second functionality is biotin and the third functionality is streptavidin. 
     
     
         32 . The kit of  claim 26 , wherein the nanoparticles are selected from gold nanoparticles, silver nanoparticles, platinum nanoparticles, magnetite nanoparticles, gold/iron alloy nanoparticles, and latex nanoparticles. 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . The kit of  claim 26 , wherein the at least one characteristic is selected from sample color, UV-VIS spectrum, UV-VIS peak wavelength, and absorbance.