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The initial purpose of this dilution is to minimize the effect of local salt concentration increase on the AuNP stability when reaction sample solution is added into AuNP solution. However, our subsequent study showed that the undiluted reaction solution could also be directly added to AuNP solution without causing significant AuNP stability change (see ESI, Fig. S2)
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The lower CIAP concentration (e.g. 0.05 units/20 μL) could also be detectable, but a longer reaction time (e.g. 12 h) was required to ensure enough ATP had been converted into adenosine to get a quick color change (1 min) in the subsequent AuNP test
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The lower CIAP concentration (e.g. 0.05 units/20 μL) could also be detectable, but a longer reaction time (e.g. 12 h) was required to ensure enough ATP had been converted into adenosine to get a quick color change (1 min) in the subsequent AuNP test
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Control experiments where the AuNP stability with or without CIAP were accessed by gradually adding NaCl solution (1 M) to AuNP solution showed that CIAP did not stabilize AuNP either under studied conditions
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Control experiments where the AuNP stability with or without CIAP were accessed by gradually adding NaCl solution (1 M) to AuNP solution showed that CIAP did not stabilize AuNP either under studied conditions
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Theoretically, there is a possibility that AuNPs are bridged by the RCA product (or H-bonds between DNA molecule-adsorbed AuNPs). However, we think this is a minor contribution to AuNP aggregation in our assay given the fact that this type of inter-particle crosslinking process is known as a relatively slow process (for example, see ref. 1a and 8c). Considering the AuNP aggregation (or color change) in our assay is completed in 1 min, we believe this aggregation is induced by the loss (or screen) of surface charges
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