Fabrication And Applicatuon Of Surface Plasmon Resonance Based Glycan Biosensors

Recently glycan biosensors have attracted a great deal of interest due to their wide applications in life science and biomedical fields,such as glycomics research,drug and vaccine screening,pathogen diagnosis,etc.DNA,lipid,proteins,and some polysaccharide can be attached on substrate easily via hydrophobic interactions or van der Waals' force.But the immobilization of small hydrophilic sugar probes(i.e.,monosaccharide and disaccharide)on surface of biosensors usually rely on either the derivation of sugar molecules with functional groups(e.g.,-SH,-NH2,etc.)or the synthesis of glycoconjugates with various scaffolds,such as dendritic polymers,DNA,lipid,neoglycoproteins and so on.Because of the multivalent nature of carbohydrate-lectin interaction,the glycan probes attached onto a biosensor surface are required to have an appropriate lateral distribution and conformation,aiming at mimicking the glycocalyx on cell surfaces,and we should control the distribution and conformation of the glycan probes.The paper demonstrated that BSA was an ideal building blocks for immobilization of sugar probes.Via the adsorption of BSA on the gold spontaneously and the mild reaction conditions of squaric acid mediated coupling,we can attach sugar probes on SPR gold substrate directly,cheaply,and easily.Immobilization of sugar probes has two strategies,one is to attach BSA-Sugar conjugates directly on the surface of gold,which is called one-step method;another is to attach the BSA on the surface of gold firstly,then via squaric acid mediated coupling reaction to attach sugar probes on BSA-coated substrate directly,which is called two-step method.Two-step method can avoid the purification of BSA-sugar conjugates.The results showed that glycan biosensors prepared by one-step method or two-step method have high affinity bindings,high specificities,and very low limits of detection.We used pseudo-first-order model and Hill equation to match the SPR kinetic measurement data of one-step method and two-step method chips,respectively.Fitting of curves were good,indicating that the corresponding models were proper.Each chip has a nanomolar equilibrium dissociation constant,which proved the multivalent effects of sugar probes with lectins.All chips could be regenerated by using 10 mM NaOH solution.The limit of detection of chips prepared by one-step method was 1.8 nM,which was 1.9 nM by two-step method.