Methods for covalent linking of optical reporters

Abstract

A method to link a light emitting reporter to biomolecules with nucleotide oligomers is described. The light reporter particles are silylated and functionalized to produce a coated light reporter particle, prior to covalently linking the biomolecules to the light reporter particle. The light reporter particle generated by the methods of the invention can be excited by a light excitation source such as UV or IR light, and when the biomolecule is DNA, the attached DNA molecule(s) are detectable by amplification techniques such as PCR.

1 - 27 . (canceled) 
     
     
         28 . A method for linking nucleic acid oligomers of detectable sequence to an optical reporter particle, the method comprising:
 introducing by covalent bonding, a plurality of reactive functional groups onto the optical reporter; and   covalently bonding the nucleic acid oligomers to the reactive functional groups.   
     
     
         29 . A method for preparing DNA labeled optical reporters of formula i
   (UCP)-[O—R 1 —X—R 2 —C(O)—NR a —R 3 -DNA] m   i
   
       wherein:
 m is an integer greater than 1; 
 X— is: —O—; —S—; or —NR b —, wherein R b  is hydrogen or C 1-6 alkyl; 
 each R a  is C 1-6 alkyl and may be the same or different at each occurrence; 
 R 1 — is C 2-8 alkylene; 
 R 2  is C 1-8 alkylene; 
 R 3  is C 2-8 alkylene; and 
 UCP is an up-converting phosphor particle 
 
       the method comprising:
 reacting activated optical reporters with a nucleic acid oligomer reagent of the formula h;
   HR a N—R 3 -DNA  h
 
 
 
       to form the DNA labeled-optical reporters of formula i. 
     
     
         30 . A method for linking nucleic acid oligomers of detectable sequence to an optical reporter particle, the method comprising:
 introducing by covalent bonding, a plurality of reactive functional groups onto the optical reporter, said optical reporter being an up-converting phosphor containing particle, said up-converting phosphor containing particle having a surface that is reactive to silylating agents,   treating the up-converting phosphor containing particle with a mixture of   
       
         
           
           
               
               
           
         
       
       wherein:
 each R is C 1-6 alkyl and may be the same or different at each occurrence; 
 R 1 — is C 2-8 alkylene; and 
 X— is: —O—; —S—; or —NR b —, wherein R b  is hydrogen or C 1-6 alkyl; 
 
       to form a plurality of groups of the formula
   —O—R 1 —XH
 
 
       covalently bound to the surface of the optical reporter,
 treating the optical reporter with a linking agent of the formula
   Y—R 2 —C(O)—OR d  
 
 
 
       wherein:
 Y is a leaving group; 
 
       R 2  is C 1-8 alkylene; and
 R d  is hydrogen or C 1-6 alkyl; 
 
       to form a plurality of groups of the formula
   —O—R 1 —X—R 2 —C(O)—OR d  
 
 
       covalently bound to the surface of the optical reporter,
 covalently bonding the nucleic acid oligomers to the reactive functional groups covalently bound to the upconverting phosphor containing particle; 
 determining at least a partial sequence of the nucleic acid oligomers covalently bound to the optical reporter; and 
 producing a composition of the formula I:
   ( c O p R)-[L-(NA)] m   I
 
 
 
       wherein:
 m is an integer greater than 1; 
 (cOpR) is a coated optical reporter particle; 
 (NA) is a nucleic acid oligomer of detectable sequence; 
 L is a linking group covalently bound to the coated optical reporter particle and to the nucleic acid oligomer. 
 
     
     
         31 . A method for linking nucleic acid oligomers of detectable sequence to an optical reporter particle, the method comprising:
 introducing by covalent bonding, a plurality of reactive functional groups onto the optical reporter;   covalently bonding the nucleic acid oligomers to the reactive functional groups; and   producing a composition of the formula I:
   ( c O p R)-[L-(NA)] m   I
 
   
       wherein:
 m is an integer greater than 1; 
 (cOpR) is a coated optical reporter particle; 
 (NA) is a nucleic acid oligomer of detectable sequence; 
 L is a linking group covalently bound to the coated optical reporter particle and to the nucleic acid oligomer. 
 
     
     
         32 . A method for producing a composition of the formula I:
   ( c O p R)-[L-(NA)] m   I
   
       wherein:
 m is an integer greater than 1; 
 (cOpR) is a coated optical reporter particle; 
 (NA) is a nucleic acid oligomer of detectable sequence; 
 L is a linking group covalently bound to the coated optical reporter particle and to the nucleic acid oligomer, 
 introducing by covalent bonding, a plurality of reactive functional groups onto the optical reporter; and 
 covalently bonding the nucleic acid oligomer to the reactive functional groups. 
 
     
     
         33 . A composition of the formula I:
   ( c O p R)-[L-(NA)] m   I
   
       wherein:
 m is an integer greater than 1; 
 (cOpR) is a coated optical reporter particle; 
 (NA) is a nucleic acid oligomer of detectable sequence; 
 L is a linking group covalently bound to the coated optical reporter particle and to the nucleic acid oligomer, 
 
     
     
         34 . The composition of  claim 33 , wherein (cOpR) comprises an upconverting phosphor (UCP) material. 
     
     
         35 . The composition of  claim 33 , wherein (NA) is a single or double stranded DNA molecule having a length of between about 40 base pairs and about 1000 base pairs. 
     
     
         36 . The composition of  claim 33 , wherein L comprises an alkylene moiety having a first end covalently bound to the coated optical reporter particle and a second end covalently bound to the nucleic acid oligomer. 
     
     
         37 . The composition of  claim 34 , wherein (UCP) is an upconverting phosphor particle of the formula:
   Y x Yb y Er z O 2 S; or     Na(Y x Yb y Er z )F 4 ;   
       wherein:
 x is from about 0.6 to about 0.95; 
 y is from about 0.05 to about 0.35; and 
 z is from about 0.1 to about 0.001. 
 
     
     
         38 . The composition of  claim 33 , wherein L is of the formula:
   -A-R 1 —B—
   
       wherein:
 R 1  is C 2-8 alkylene; 
 -A- is a group covalently bonded to the surface of the coated optical reporter; and 
 —B— is a group covalently bonded to the 3′ or 5′ end of the nucleic acid oligomer. 
 
     
     
         39 . The composition of  claim 38 , wherein -A- is —O—. 
     
     
         40 . The composition of  claim 38 , wherein —R 1 — is —(CH 2 ) n — and wherein n is from 2 to 8. 
     
     
         41 . The composition of  claim 38 , wherein —B— is:
 —S—; 
 —O—; 
 —NR a —; 
 —S—(CH 2 ) p —; 
 —O—(CH 2 ) p —; 
 —NR a —(CH 2 ) p —; 
 —S—(CH 2 ) q —C(O)—NR a —(CH 2 ) p —; 
 —O—(CH 2 ) q —C(O)—NR a —(CH 2 ) p —; 
 —NR a —(CH 2 ) q —C(O)—NR a —(CH 2 ) p —; 
 —S—C(O)—(CH 2 ) r —C(O)—NR a —(CH 2 ) p —; 
 —O—C(O)—(CH 2 ) r —C(O)—NR a —(CH 2 ) p —; or 
 —NR a —C(O)—(CH 2 ) r —C(O)—NR a —(CH 2 ) p —; 
 
       wherein:
 p is from 2 to 8; 
 q is from 1 to 8; 
 r is from 2 to 8; and 
 each R a  is independently hydrogen or C 1-6 alkyl; 
 
     
     
         42 . The composition of  claim 38 , wherein —B— is:
 —S—(CH 2 ) q —C(O)—NR a —(CH 2 ) p  or 
 —NR a —C(O)—(CH 2 ) r —C(O)—NR a —(CH 2 ) p —; 
 
       wherein p, q, r and R a  are as recited in  claim 41 . 
     
     
         43 . The composition of  claim 42 , wherein:
 p is from 2 to 6;   q is from 1 to 3; and   r is 2 or 3.   
     
     
         44 . The composition of  claim 38 , wherein —B— is:
 —S—CH 2 —C(O)—NH—(CH 2 ) 6 —; or 
 —NH—C(O)—(CH 2 ) 3 —C(O)—NH—(CH 2 ) 6 —; 
 
     
     
         45 . The composition of  claim 33 —Please double check this, wherein the cOpR is coated with silica. 
     
     
         46 . The composition of  claim 45 , wherein the silica comprises at least one Si—O bond. 
     
     
         47 . A composition of the formula II:
   (UCP)-[A-R 1 —X—R 2 —C(O)—NR a —R 3 -(DNA)] m   II
   wherein:   m is an integer greater than 1;   UCP is an upconverting phosphor particle;   DNA is a single or double stranded deoxyribonucleic acid oligomer;   -A- is a group capable of covalently bonding to the surface of the Upconverting phosphor particle;   R 1  is C 2-8 alkylene,   R 2  is C 1-8 alkylene or —C(O)—C 1-8 alkylene-;   —X— is —O—, —S— or —NR a —;   R 3  is C 2-8 alkylene; and   R a  is hydrogen or C 1-6 alkyl.   
     
     
         48 . The composition of  claim 47 , wherein the composition is of the formula IV:
   (UCP)-[O—(CH 2 ) s —S—(CH 2 ) t —C(O)—NH—(CH 2 ) v -(DNA)] m   IV
   
       wherein:
 s is from 2 to 6; 
 v is from 2 to 6; 
 t is from 1 to 3; and 
 m, UCP and DNA are as recited in  claim 47 . 
 
     
     
         49 . The composition of  claim 47 , wherein the composition is of the formula V:
   (UCP)[O—(CH 2 ) s —NH—C(O)—(CH 2 ) u —C(O)—NH—(CH 2 ) v -(DNA)] m   V
   
       wherein:
 s is from 2 to 6; 
 v is from 2 to 6; 
 u is 2 or 3; and 
 m, UCP and DNA are as recited in  claim 47 . 
 
     
     
         50 . The composition of  claim 47 , wherein the composition is of the formula VI:
   (UCP)[O—(CH 2 ) 3 —S—CH 2 —C(O)—NH—(CH 2 ) 6 -(DNA)] m   VI
   
       wherein m, UCP and DNA are as recited in  claim 47 . 
     
     
         51 . The composition of  claim 47 , wherein the composition is of the formula VII:
   (UCP)-[O—(CH 2 ) 3 —NH—C(O)—(CH 2 ) 3 —C(O)—NH—(CH 2 ) 6 -(DNA)] m   VII
   
       wherein m, UCP and DNA are as recited in  claim 47 . 
     
     
         52 . The composition of  claim 33 , wherein the cOpR comprises a visually detectable light emitting material selected from the group consisting of a fluorescent dye, a upconverting phosphor, a ceramic powder, or a quantum dot. 
     
     
         53 . The composition of  claim 52 , where said light emitting materials are excitable by UV or an infrared light source. 
     
     
         54 . The composition of  claim 33 , wherein the cOpR comprises at least one electromagnetic emitting material. 
     
     
         55 . The composition of  claim 54 , where the electromagnetic emitting material is detectable by devices which provide sources selected from the group consisting of an infrared radiation source, magnetic field source, a quantum dot or electromagnetic pulse.