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A simple two-step protocol for modification of atomic force microscopy (AFM) tip and substrate by using a "click reaction" has been developed. The modified tip and substrate would be applied to detect trace amounts of ricin by using atomic force microscopy. A key feature of the approach is the use of a PEG (polyethylene glycol) derivative functionalized with one thiol and one azide ending group. One end of the PEG was attached to the gold-coated AFM tip by a strong Au-thiol bond. The azide group hanging at the other end of the immobilized PEG was used for the attachment of an antiricin antibody modified with an alkyne group using a "click reaction". The latter reaction is highly efficient, compatible with the presence of many functional groups and could proceed under mild reaction conditions. In a separate step, ricin was immobilized on the gold substrate surface that was modified by active esters. For this process, a novel bifunctional reagent was employed containing an active ester and a thioctic acid moiety. By these modification processes, AFM recognition imaging was used to detect the toxin molecules and the results show fg/mL detection sensitivity, surpassing the existing detection techniques. With measurement of the unbinding force between the antiricin antibody and ricin, which was statistically determined to be 64.89 +/- 1.67 pN, the single molecular specificity of this sensing technique is realized.

Simple, Clickable Protocol for Atomic Force Microscopy Tip Modification and Its Application for Trace Ricin Detection by Recognition Imaging