Method of fabrication of photonic biosensor arrays

Abstract

This invention relates to a method for the fabrication of photonic biosensor arrays and applications of arrays produced by the method in the biomedical field. A method for the fabrication of a biosensor array for plasmon resonance-based sensing of a plurality of different biological targets simultaneously, the method comprising: (i) providing a transparent substrate; ii) providing seed metallic nanoparticles in the form of a colloid; (iii) depositing said colloid as discrete metallic islands on the transparent substrate, each of said metallic islands comprising a plurality of metallic nanoparticles; (iv) washing the substrate in order to remove unadhered material; (v) developing the substrate in a growth solution, which solution comprises a salt of the same metal which is present in the form of discrete metallic islands on the substrate, a reducing agent, a capping agent and optionally a surfactant; (vi) washing the developed substrate; and (vii) functionalising each of said metallic islands with a different functionalising molecule using a common chemical process to attach said different functionalising molecules to said metallic islands.

1 .- 28 . (canceled) 
     
     
         29 . A method for the fabrication of a biosensor array for plasmon resonance-based sensing of a plurality of different biological targets simultaneously, the method comprising:
 (i) providing a transparent substrate;   (ii) providing seed metallic nanoparticles in the form of a colloid;   (iii) depositing said colloid as discrete metallic islands on the transparent substrate, each of said metallic islands comprising a plurality of metallic nanoparticles;   (iv) washing the substrate in order to remove unadhered material;   (v) developing the substrate in a growth solution, which solution comprises a salt of the same metal which is present in the form of discrete metallic islands on the substrate, a reducing agent, a capping agent and optionally a surfactant;   (vi) washing the developed substrate; and   (vii) functionalising each of said metallic islands with a different functionalising molecule using a common chemical process to attach said different functionalising molecules to said metallic islands.   
     
     
         30 . A method as claimed in  claim 29 , wherein the substrate comprises an uncoated silica/glass surface. 
     
     
         31 . A method as claimed in  claim 29 , wherein the metallic nanoparticles comprise copper, silver, gold, platinum, palladium and iridium or a mixture or an alloy thereof. 
     
     
         32 . A method as claimed in  claim 29 , wherein, in step (ii), the seed metallic nanoparticles provided in the form of a colloid are substantially spherical with a diameter in the range of from 2 to 6 nm. 
     
     
         33 . A method as claimed in  claim 29 , wherein, in development step (v), the growth solution comprises a metal salt, a reducing agent, a capping agent and a surfactant. 
     
     
         34 . A method as claimed in  claim 29 , wherein, in development step (v), the growth solution comprises a metal salt, a reducing agent, and the capping agent cetyltrimethylammonium bromide (CTAB). 
     
     
         35 . A method as claimed in  claim 29  wherein said metallic nanoparticles comprise gold nanoparticles. 
     
     
         36 . A method as claimed in  claim 35 , wherein, in the development step (v), the gold salt comprises HAuCl 4  or a potassium, calcium, sodium, or lithium salt thereof. 
     
     
         37 . A method as claimed in  claim 35 , wherein, in the development step (v), the capping agent is CTAB and the reducing agent is ascorbic acid. 
     
     
         38 . A method as claimed in  claim 29 , wherein functionalising step (vii) comprises using a solution deposition head with a plurality of nozzles to collect a plurality of said different functionalising molecules from a plurality of reservoirs and to deposit said plurality of functionalising molecules onto a respective plurality of said metallic islands. 
     
     
         39 . A method as claimed in  claim 38 , wherein said different functionalising molecules are attached to said metallic nanoparticles using the same ligand. 
     
     
         40 . A method as claimed in  claim 38 , wherein, in functionalising step (vii), a fraction of the respective plurality of said metallic islands are left un-functionalised and instead comprise a plurality of control spots. 
     
     
         41 . A method for controlling the size distribution of nanoparticles grown in accordance with the following steps:
 (i) provision of a transparent substrate;   (ii) provision of seed metallic nanoparticles in the form of a colloid;   (iii) deposition of said colloid as discrete metallic islands on the transparent substrate, each of said metallic islands comprising a plurality of metallic nanoparticles;   (iv) washing of the substrate in order to remove unadhered material;   (v) development of the substrate in a growth solution, which solution comprises a salt of the same metal which is present in the form of discrete metallic islands on the substrate, a reducing agent, a capping agent and optionally a surfactant;   which method comprises adding metal ions to the growth solution of development step (v), wherein these additional metal ions are different to the metal forming the plurality of metallic islands on the substrate.   
     
     
         42 . A method as claimed in  claim 41 , which comprises the further steps of (vi) washing; and (vii) functionalisation of each of said metallic islands with a different functionalising molecule using a common chemical process to attach said different functionalising molecules to said metallic islands. 
     
     
         43 . A biosensor array for plasmon resonance-based sensing of a plurality of different biological targets, the array obtained by or obtainable from the fabrication method of  claim 29 . 
     
     
         44 . A biosensor array as claimed in  claim 43  comprising a transparent substrate having a surface bearing a plurality of array spots for plasmon resonance sensing, each of said array spots comprising a discrete metallic island to which is attached functionalising molecules for binding to a biological target, different said islands bearing different said functionalising molecules for binding to different ones of said biological targets, and wherein total internal reflection of light at said surface at a wavelength at or near a said plasmon resonance results in scattering of said light away from said surface, said scattering being modulated by said binding of said biological targets. 
     
     
         45 . A biosensor array as claimed in  claim 43  wherein said metallic nanoparticles have at least one dimension of less than 30 nm. 
     
     
         46 . A biosensor array as claimed in  claim 43  wherein said nanoparticles comprise rod-like nanoparticles. 
     
     
         47 . A biosensor array as claimed in  claim 43  wherein said nanoparticles include nanoparticles forming an optical antenna for said light wherein said optical antenna comprises an adjacent pair of nanoparticles having a generally rod-like or triangular shape, and having adjacent ends separated by a gap of less than 100 nm, preferably less than 50 nm, said nanoparticles having physical lengths which are resonant for a said plasmon resonance at substantially the same optical wavelength. 
     
     
         48 . A biosensor array as claimed in  claim 43  further comprising a plurality of control spots, a said control spot substantially lacking said functionalising molecules. 
     
     
         49 . A biosensor array as claimed in  claim 48  wherein a said control spot is capable of detecting a change in the bulk refractive index (Δn) of the order of 1×10 −5  or lower refractive index units (RIU). 
     
     
         50 . A biosensor array as claimed in  claim 43  wherein said different functionalising molecules are attached to said metallic nanoparticles using the same ligand. 
     
     
         51 . A biosensor array as claimed in  claim 43  wherein said metallic nanoparticles comprise gold nanoparticles. 
     
     
         52 . A method of plasmon resonance-based sensing of a plurality of different biological targets simultaneously, the method comprising:
 coupling light of at least one wavelength into a biosensor array as claimed in claim  15  such that total internal reflection of said light at said surface generates an evanescent wave field which excites plasmons in said functionalised metallic nanoparticles and scatters said light;   flowing a fluid carrying a plurality of said biological targets for analysis over said array;   imaging said scattered light from said array to generate image data for said biological targets carried by said fluid; and   analysing said image data to determine levels of said biological targets carried by said fluid.   
     
     
         53 . A method as claimed in  claim 52  wherein said imaging is performed in real-time to follow binding kinetics of said biological targets carried by said fluid, and wherein said analysing comprises determining time variations of binding of said different targets to said different functionalising molecules. 
     
     
         54 . A method of analysing a plurality of different biological targets simultaneously, the method comprising:
 fabricating an array according to the method of claim  1 ;   passing a fluid carrying a plurality of said biological targets for analysis over said array; and   detecting said biological targets carried by said fluid by imaging light scattered by plasmon resonance at said functionalised metallic islands.