Functionalized biochips for spr-ms coupling

Abstract

The invention relates to a method for coupling in-line the analysis of molecular interactions by surface plasmon resonance (SPR) with a structural identification by mass spectrometry using the same functionalized support for both types of analysis.

1 . A method for functionalizing the metal face of a support for analysis by surface plasmon resonance, said method comprising grafting a self-assembled monolayer of poly(ethylene oxide) directly onto the metal face of said support,
 in which the metal of the metal face is gold, and   in which the poly(ethylene oxide) is a compound of formula (I)
   A-(CH 2 ) n —(O—CH 2 —CH 2 ) x -D  (I) 
   in which:
 n is equal to 1 or 2; 
 x is an integer between 5 and 16; 
 A is a group for anchoring the PEO onto the gold surface of the support by a strong interaction; and 
 D is an optionally modified group for the binding of biomolecules. 
   
     
     
         2 . The method according to  claim 1 , in which n is equal to 2. 
     
     
         3 . The method according to  claim 1 , in which x is equal to 8. 
     
     
         4 . The method according to  claim 1 , in which A corresponds to an —SH group. 
     
     
         5 . The method according to  claim 1 , in which the PEO is O-(2-mercaptoethyl)-O′-(2-carboxyethy)heptaethylene glycol of formula HS—CH 2 —CH 2 —(O—CH 2 —CH 2 ) 8 —COOH. 
     
     
         6 . The method according to  claim 1 , which method comprises the series of following steps:
 1) prior cleaning of the support;   2) grafting of the PEO onto the support; and   3) optionally, modification of group D of the PEO.   
     
     
         7 . The method according to  claim 6 , in which the cleaning is carried out by UV-ozone treatment. 
     
     
         8 . The method according to  claim 6 , in which group D represents a —COOH group which is modified in step 3) so as to give an N-hydroxysuccinimide group. 
     
     
         9 . The method according to  claim 6 , in which the grafting is carried out by immersing the support in a vessel containing the poly(ethylene oxide) to be grafted, in solution. 
     
     
         10 . A functionalized support for analysis by surface plasmon resonance, comprising a metal face onto which a self-assembled monolayer of poly(ethylene oxide) is directly grafted,
 in which the metal face is made of gold, and   in which the poly(ethylene oxide) is a compound of formula (I)
   A-(CH 2 ) n —(O—CH 2 —CH 2 ) x -D  (I) 
   in which:
 n is equal to 1 or 2; 
 x is an integer between 5 and 16; 
 A is a group for anchoring the PEO onto the gold surface of the support by a strong interaction; and 
 D is an optionally modified group for the binding of biomolecules. 
   
     
     
         11 . The functionalized support produced by the method according to  claim 1 . 
     
     
         12 . The functionalized support according to  claim 10 , further comprising “receptor” molecules immobilized on said support via a bond by means of grafted poly(ethylene oxide). 
     
     
         13 . A method of surface plasmon resonance comprising a functionalized support according to  claim 10 , in particular in an surface plasmon resonance imaging (SPRi) experiment. 
     
     
         14 . The method according to  claim 13 , said method comprising linking “receptor” molecules to the surface of said support and studying the molecular interactions of these molecules by surface plasmon resonance, in particular by SPRi. 
     
     
         15 . A method of mass spectrometry comprising a functionalized support according to  claim 10 , in order to structurally identify analytes which have been specifically retained by receptor molecules covalently linked to said functionalized support. 
     
     
         16 . The method according to  claim 15 , in which the mass spectrometry is carried out with MALDI-type ionization. 
     
     
         17 . The method of a functionalized support according to  claim 11 , for carrying out two consecutive analyses:
 an analysis by SPR; then   an analysis by mass spectrometry.   
     
     
         18 . The method according to  claim 17 , in which the analysis by surface plasmon resonance is an analysis by surface plasmon resonance imaging. 
     
     
         19 . The method according to  claim 17 , in which the analysis by MS is an analysis of MALDI type, in particular an analysis of MALDI-TOF type. 
     
     
         20 . A method for coupling an analysis by SPR, in particular SPRi, with an analysis by MALDI MS or MS/MS, comprising:
 1) immobilization of one or more “receptor” molecules on a functionalized support according to  claim 10 ; then   2) placing of the support in an SPR analyzer and analysis, by SPR, of the interactions between the “receptor” molecule(s) immobilized and a sample of analytes; then   3) removal of the support from the SPR analyzer and placing of said support in a mass spectrometer, and structural analysis, by MS, of the analytes specifically retained by the “receptor” molecules during the SPR analysis.   
     
     
         21 . A method for coupling an analysis by SPR, in particular SPRi, with an analysis by MS, in particular MALDI MS, or MS/MS comprising:
 1) immobilization of one or more “receptor” molecules on a functionalized support according to  claim 10 ; then   2) placing of the support in an SPR analyzer and an analysis, by SPR, of the interactions between the “receptor” molecule(s) immobilized and a sample of analytes;   3) in situ localized enzyme digestion of the analytes retained on the spots of the “receptor” molecules immobilized on the functionalized support, then placing of the support in a mass spectrometer, and structural analysis, by MS, or MS/MS, of the products of digestion of the analyte(s) present on the functionalized support.   
     
     
         22 . A method for coupling an analysis by SPR, in particular SPRi, with an analysis by MS, in particular MALDI MS, comprising:
 1) immobilization of one or more “receptor” molecules on a functionalized support according to  claim 10 ; then   2) placing of the support in an SPR analyzer and analysis, by SPR, of the interactions between the “receptor” molecule(s) immobilized and a sample of analytes; then   3) removal of the support from the SPR analyzer and placing of said support in a mass spectrometer, and structural analysis, by MS, of the analytes specifically retained by the “receptor” molecules during the SPR analysis; then   4) in situ localized enzyme digestion of the analytes retained on the spots of the “receptor” molecules immobilized on the functionalized support, and placing of the support in a mass spectrometer, and structural analysis, by MALDI MS or MALDI MS/MS, of the products of digestion of the molecule(s) present on the functionalized support.