Specific Versus Non‐specific Response in Exponential Molecular Amplification from Cross‐Catalysis: Modeling the Influence of Background Amplifications on the Analytical Performances

Abstract

Molecular based signal amplifications relying on an autocatalytic process may represent an ideal strategy for the development of ultrasensitive analytical or bioanalytical assays, the main reason being the exponential nature of the amplification. However, to take fully advantage of such amplification rates, high stability of the starting co-reactants is required in order to avoid any undesirable background amplification. Here, on the basis of a simple kinetic model of cross-catalysis including a certain degree of intrinsic instability of co-reactants, we highlight the key parameters governing the analytical response of the system and discuss the analytical performances that are expected from a given kinetic set. In particular, we show how the detection limit is directly related to the relative instability of reactants within each catalytic loop. The model is validated with an experimental dataset and is intended to serve as a guide in the design and optimization of autocatalytic molecularbased amplification systems with improved analytical performances.

Exponential amplification by redox cross-catalysis and unmasking of doubly protected molecular probes

“…Improvement of the rates associated to each loop of the cross-catalysis reaction is a strategy to shorten the analysis time and to potentially enhance the detection limit by better discriminating the specific from nonspecific responses. 36 In the present case, the background amplification stems from the instability associated to loop 2 ( i.e. , from NADH autoxidation or NADH-oxidase like activity of diaphorase) since the double masking of the probe makes it so stable that no nonspecific response can arise from loop 1.…”

“…, from 0.5 to 20 µM H 2 O 2 ) confirms the autocatalytic nature of the reaction. The deviation from linearity observed at the lowest concentrations (with a tendency to merge progressively with the t 1/2 value determined in the absence of H 2 O 2 ) illustrates the limitation imposed by the nonspecific response on the analytical performances, 36 leading here to a LoD of only 0.2 µM H 2 O 2 , a value which is finally not much different from that we had previously obtained with the self-immolating probe P 1 under similar experimental conditions. 34 Nevertheless, the kinetics with P 2 are much faster than previously (by a factor 6), which is clearly an advantage to shorten the analysis time.…”

“… 34 Unfortunately, no gain in LoD was obtained, suggesting a relatively similar pH dependence of both probe reactivity and stability. 36 …”

“…This asymmetry in the analytical performances was shown to be an intrinsic property of a cross-catalytic reaction scheme. 36 Indeed, if the two catalytic loops have similar rates, the detection efficiency of the catalyst of loop 1 or loop 2 is theoretically the same, thus leading to an identical LoD of each trigger. On the other hand, if the rates of each loop are different, the trigger of the fastest loop becomes the most easily detectable and therefore the one that theoretically leads to the lowest LoD.…”

“… 34 Another important feature we have considered here is to design boronate probes with an improved reactivity towards H 2 O 2 as well as an optimized redox cycling capability, with the underlying idea to outcompete the background reactions. 36 …”