the details of embodiments of the present disclosure have been set forth in the accompanying description below . although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure , the preferred methods and materials are now described , other features , objects , and advantages of the present disclosure will be apparent from the description and from the claims . in the specification and the appended claims , the singular forms include plural references unless the context clearly dictates otherwise . all patents and publications cited in this specification are incorporated by reference in their entirety . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present disclosure pertain . streptavidin interacts with other small molecules such as adenosine , which may result in non - specific interactions in the presence of unoccupied biotin binding sites . ( see t . bing et al ., bioarg med chem lett , 2012 , 22 , 7052 - 7055 ). the four high - affinity binding sites of streptavidin can bind multiple biotinylated ligands and cause target aggregation . however , because protein aggregation can change its biological functions , the use of streptavidin in live cells can be prone to artifacts . the doubly bound form of streptavidin ( sa - b 2 ) is required to form a 2d crystal on a biotin bearing lipid monolayer . ( see m . fukuto et al ., soft matter , 2010 , 6 , 1513 ). a class of three - arm bisbiotin ligands , ( 5a ) and ( 5b ), are synthesized as outlined in fig1 . in these reactions , reagents and conditions are as follows : ( i ) pd ( pph3 ) cl2 , cui , the et3n = 1 : 1 , rt , 5 h ; ( ii ) s7a or s7b , pyridine , dichloromethane , rt , 12 h ; ( iii ) trifluoroacetic acid , rt , 10 min ; ( iv ) biotin n - hydroxysuccinimide ester , triethylamine , dmf , rt , 3 h . the synthesis of starting materials s7a and s7b used in an intermediate reaction of the synthesis shown in fig1 is shown in fig2 , fig3 shows a y shaped conformation of bisbiotin ligand ( 5a ) generated from computation software spartan ′ 14 ( wavefunction inc .). first , the 2d chemical structures were drawn in chembiodraw ultra 11 and exported to spartan ′ 14 to generate a 3d structure that was subjected to energy minimization using the built - in mmff94s molecular mechanics , and then the structure was optimized using the dft ( density functional theory ) calculation , b3lyp with basis set 6 - 31 + g * in vacuum . the structure of conjugate ( 6 ), a three - arm bisbiotin ligand ( 5a ) conjugated with a fluorescent dye dbco - fluor 585 ( from kerafast ®) is shown in fig4 . a conjugate ( 7 ) of mono - biotin with dbco - fluor 585 was also synthesized and used as a positive control ( see fig5 ). fig6 is an image of gel electrophoresis of streptavidin products interacting with the fluorescent biotin ligands ( 6 ) and ( 7 ). in the image , lane 1 shows streptavidin incubated with fluorescently tagged mono - biotin ligand 7 in a 1 : 1 ratio at room temperature ; lane 2 shows streptavidin incubated with fluorescently tagged bisbiotin ligand 6 at room temperature ; lane 3 shows streptavidin with fluorescent dye ( dbco - fluor 585 negative control ); and lane 4 shows streptavidin alone . the bands are located at the position where a 53 kd molecular mass standard migrates to by gel electrophoresis . as indicated by the 53 kd band in lane 2 , the bisbiotin conjugate ligand ( compound ( 6 ) of fig4 ) can form a 1 : 1 complex with streptavidin . in fig7 a and 7b show the images from gel electrophoresis analysis of streptavidin and its complexes with mono biotin ligand ( compound ( 8 ), shown in fig8 ) and bisbiotin ligand ( 5a ) ( fig1 ), which were incubated at different temperatures . in fig7 a , streptavidin alone was incubated at room temperature ( rt ), 35 ° c ., 45 ° c ., 55 ° c ., 70 ° c ., 85 ° c ., and 95 ° c . streptavidin is thermally stable up to 45 ° c . ( the dark band corresponds to a 53 kd tetrameric streptavidin ). at 55 ° c ., a light band appears corresponding to 13 kd monomeric streptavidin . at 70 ° c ., the intensity of the monomer band increases significantly . at the higher temperature ( 85 ° c . ), the tetranieric streptavidin is completely disintegrated into its monomers . fig7 b demonstrates that the thermal stability of streptavidin is increased by formation of complexes with biotins . when the streptavidin forms a 1 : 2 complex with the mono - biotin ligand ( 8 ) ( structure shown in fig8 ), indicated as “ linear linker ”, streptavidin disintegration is reduced significantly at 70 ° c . however , the streptavidin is disintegrated completely at 85 ° c . importantly , there was a distinct difference in thermal stability of the streptavidin tetramer between a linear linker complex ( compound ( 8 ) incubated with streptavidin ) and the y - shaped trilinker complex ( in this example , compound ( 5a ) incubated with streptavidin ). when the streptavidin forms a 1 : 1 complex with the bisbiotin ligand ( 5a ) ( shown in fig1 ) ( equal to 2 equivalents of compound ( 8 ) in the number of effective biotin moieties ), its complete disintegration takes place at 95 ° c . based on the gel electrophoresis , indicated as “ trilinker ” in fig7 b . thus , the bisbiotin ligands of the instant disclosure are more thermostable than the mono - biotin ligands . atomic force microscopy ( afm ) can be used to measure the mechanical force necessary to break the biotin - streptavidin interactions . the bisbiotin ligand ( 5b ) is attached to an afm tip through the method disclosed in u . s . provisional application ser . no . 61 / 898 , 177 . the attachment of biotin compounds of the present disclosure to afm tip may include a peg , or any other suitable linker known in the art . streptavidin molecules are immobilized onto a mica substrate by a known method ( see h . wang et al ., biophysical j ., 2002 , 83 , 3619 - 3625 ) immobilization of bisbiotin ligands on an afm tip can be utilized for attaching streptavidin followed by biotinylated antibodies and other affinity molecules . fig9 shows a representative force distance curve ( dotted lines ) of the bisbiotin ligand ( 5b ) unbinding from the streptavidin immobilized on the mica substrate . as shown in this figure , the unbinding force of ( 5b ) ( see the rupture at the distance of ˜ 13 nm ) is larger than that of the mono - biotin ligand ( solid line ). in fact , the force histograms show that the mono biotin ligand has a force peak around ˜ 68 . 5 pn ( see fig1 a ) and the bisbiotin ligand has two force peaks around ˜ 70 pn and ˜ 131 pn ( see fig1 b ). accordingly , this study shows that the interaction of streptavidin with bisbiotin is much stronger than that with mono - biotin . the bisbiotin ligands of the present disclosure can be immobilized on a solid surface . to demonstrate the relative utility of such immobilized mono and bisbiotin compounds , thiolated compounds were synthesized , as shown in fig1 and 12 , immobilized on gold substrates , and subjected to surface plasmon resonance ( spr ). fig1 shows the synthesis of a thiolated mono - biotin ligand ( 10 ). in the synthesis , biotin 2 -( diphenylphosphino ) phenolester ( 9 ) is synthesized as a precursor for reacting with azido molecules , for example , azido - peg - alkyl - sac . fig1 shows the synthesis of a thiolated bisbiotin ligand ( 12 ). reacting the bisbiotin ligand ( 5a ) with 10 -( acetylthio ) decanoic acid 2 -( diphenylphosphino ) phenol ester ( 11 ) yields a thiolated bisbiotin ligand ( 12 ). mixed monolayers of the mono and bisbiotin compounds can be formed on gold substrates , as illustrated in fig1 a and 13b . the mono - biotin ligand ( 10 ) forms mixed monolayers on a gold substrate in the presence of pyrrolidine , as illustrated in fig1 a , and the bisbiotin ligand ( 12 ) is used to form a mixed monolayer on a gold substrate in the presence of pyrrolidine as illustrate in fig1 b . in one embodiment , a streptavidin layer is quickly formed after injecting a streptavidin solution onto the mono - biotin monolayer on a gold substrate through a surface plasmon resonance ( spr ) flow cell . in fig1 , the spr sensorgram shows that 74 % of streptavidin is sharply dissociated from the surface after injecting a free biotin solution in a period of 27 minutes . in another embodiment , a streptavidin layer is quickly formed after injecting a streptavidin solution onto the bisbiotin monolayer on a gold substrate through a spr flow cell , in fig1 , the spr sensorgram shows that 15 % of streptavidin is dissociated very slowly after injecting the same amount of free biotin solution . this exemplary data demonstrates the higher relative stability of bisbiotin bound streptavidin compared to mono - biotin bound streptavidin . the increased stability of the bisbiotin ligands of the present disclosure complexed to streptavidin provides the potential utility of the bisbiotin compounds of the present disclosure in assays and arrays that would benefit from immobilization on a substrate or film . biotinylated dna probes and antibodies can be attached to streptavidin that is immobilized through the bisbiotin on a solid surface without the streptavidin dissociating form the surface . likewise , any assays that have been developed using mono - biotin binding streptavidin may be improved by utilizing the bisbiotin compounds of the present disclosure . such bisbiotin compounds in complex with streptavidin demonstrate increased thermal stability and slowed dissociation , as well as the ability to withstand greater mechanical force compared to prior art mono - biotin - streptavidin complexes . in fact , no other bisbiotin ligands reported in the literature have demonstrated the same thermal stability as that observed with the bisbiotin ligands described herein . moreover , the unique three - arm structure of the bisbiotin ligands described herein allows the connection of biomolecules to solid surfaces for detection , separation , and labelling through streptavidin binding , for example , embodiments of the disclosure demonstrate , for the first time , the use of a bisbiotin ligand to immobilize streptavidin on a gold substrate . the details of one or more embodiments of the present disclosure are set forth in the accompanying description above and has been presented only for the purposes of illustration and is not intended to limit embodiments and inventions disclosed herein to the precise form disclosed . any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure . still other embodiments of the present disclosure may be patentable over prior art references for expressly lacking one or more elements disclosed in the prior art ( i . e ., claims covering such embodiments may include negative limitations ). other features , objects , and advantages of embodiments of the present disclosure will be apparent from the description and from the originally filed claims ( as well as claims supported by the present disclosure ). in the specification and the appended claims , the singular forms include plural referents unless the context clearly dictates otherwise . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present disclosure belong . all patents and publications cited in this specification are incorporated by reference .