Extended Data Fig. 4: Fluorescence spikes only occur when there is a hydrolysis reaction and when the nanoantennas are close to the enzyme.

(a) Typical nanoantenna fluorescence signal used to monitor pNPP hydrolysis. (b) Addition of the reaction products, p-nitrophenol (pNP) and inorganic phosphate (P_i), does not give a fluorescence spike because there is no hydrolysis reaction. (c) Using an enzyme without phosphatase activity and which will not hydrolyze pNPP (for example, biotinylated glucose oxidase, bGOx), does not give a fluorescence spike because there is no hydrolysis reaction. (d) Here, the ‘Dummy’ nanoantenna does not have the dye (that is, no FAM) but it is still attached to SA via its biotin, while the ‘Global’ nanoantenna has FAM but it is not biotinylated and instead is free in solution. Thus, the hydrolysis reaction of pNPP still occurs, but this system does not monitor it since there is essentially no FAM-bAP interaction. (e) Here, 3′-thiolated L12 ssDNA nanoantennas were covalently attached to the lysine residues of AP (NA-AP; see Online Methods section). This nanoantenna-AP conjugate displays a spike during pNPP hydrolysis similar to the nanoantenna-SA-bAP complex. Although several synthesis steps are involved, it may be desirable for applications for which one does not wish to use the biotin-streptavidin platform. Note that the power was reduced from 635 V to 450 V due to the high baseline. (f) As a control, unattached thiolated nanoantennas and unconjugated AP do not display a spike during pNPP hydrolysis. (g) Here, a commercially prepared conjugate of SA covalently attached to AP (SA-AP) was used. The kinetic signature is shown for the PolyT L24 nanoantenna binding to SA-AP that results in fluorescence quenching, followed by pNPP hydrolysis that results in a spike. (h) Without knowledge of the SA-AP molecular weight due to an unknown number of conjugated SAs added by the manufacturer, we instead optimized using SA-AP volume (1, 2, 3, 4, 5, 7 and 14 μL SA-AP). All experiments were performed with n=1 biologically independent enzyme samples examined over 3 independent experiments near the apparent maximum (2 to 5 μL) and 1 otherwise. Data are presented as mean values ± SEM. Even after this simple optimization, however, the spike intensity during pNPP hydrolysis remains weaker compared to using the SA and bAP strategy. Overall, these results show that no matter which attachment strategy is used, and despite some being better than others, FAM will still find its binding site on the AP enzyme. Conditions: (a-d) 150 nM L12 PolyT nanoantenna, 50 nM SA, 150 nM bAP, (e,f) ~40 nM nanoantenna-bAP conjugate and (g,h) 150 nM nanoantenna, 1 to 14 μL SA-AP; 100 µM pNPP in 200 mM Tris, 300 mM NaCl, 1 mM MgCl₂, pH 7.0, 37 °C.