Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and non-intercalating redox probes

Abstract

We present a comparative study of ten redox-active probes for use in real-time electrochemical loop-mediated isothermal amplification (LAMP). Our main objectives were to establish the criteria that need to be fulfilled for minimizing some of the current limitations of the technique and to provide future guidelines in the search for ideal redox reporters. To ensure a reliable comparative study, each redox probe was tested under similar conditions using the same LAMP reaction and the same entirely automatized custom-made real-time electrochemical device (designed for electrochemically monitoring in real-time and in parallel up to 48 LAMP samples). Electrochemical melt curve analyses were recorded immediately at the end of each LAMP reaction. Our results show that there are a number of intercalating and non-intercalating redox compounds suitable for real-time electrochemical LAMP and that the best candidates are those able to intercalate strongly into ds-DNA but not too much to avoid inhibition of the LAMP reaction. The strongest intercalating redox probes were finally shown to provide higher LAMP sensitivity, speed, greater signal amplitude, and cleaner-cut DNA melting curves than the non-intercalating molecules.

Ultimate Single-Copy DNA Detection Using Real-Time Electrochemical LAMP

“…The M13mp18 and Flavobacterium columnare LAMP assays were performed with each of four different redox reporters (Scheme 1), which were selected on the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 basis of our recent screening study of DNA redox probes in real-time electrochemical LAMP. 22 Accordingly, the three DNA-intercalating ) are at pH 8.8 and vs. Ag/AgCl. compounds we selected for the present study, i.e., [Os(bpy) 2 dppz] 2+ , PhP and MB, have ds-DNA binding strengths ranging over two orders of magnitude (i.e., from ca.…”

“…osmium complex is analogous to our previously reported real-time electrochemical HDA experiments, in which baseline drift was attributed to a slow adsorption of metal complex on the working carbon electrode surface. 12,22 The higher baseline stabilities observed in the case of MB, PhP and Ru(NH 3 ) 6 3+ are advantageous for more reliably discriminating the onset-signal decreases of positive samples from the unchanged signal responses of negative controls. Baseline correction by subtraction of an extrapolated least-square fitted line across the early amplification time (considering only the early linear part of the plot) allowed us to recover the t t value for all of the positive amplification curves (t t was defined as the time required for the signal to reach a threshold decrease that corresponds to 5-fold the noise determined for the early steady-state response, i.e.…”

“…This process has been shown to occur significantly only when millimolar levels of pyrophosphate are present in Ru(NH 3 ) 6 3+ solutions. 22 It is thus necessary to wait until nearly the end of the LAMP reaction before reaching a sufficiently high pyrophosphate concentration, the consequence of which is a lowering of the detection sensitivity of Ru(NH 3 ) 6 3+ LAMP compared to the intercalating redox probes. Concerning the shape of amplification curves, the DNA intercalator [Os(bpy) 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 reporter.…”

“…21 For the latter, it was initially assumed that the electrochemical signal decrease resulted from the electrostatic interaction between Ru(NH 3 ) 6 3+ and the anionic DNA backbone of amplicons, 21 but we have very recently demonstrated that this was instead the consequence of a coprecipitation of Ru(NH 3 ) 6 3+ with the pyrophosphate anions generated during LAMP. 22 With this nonintercalating redox probe, a detection limit of 20-30 target DNA copies/µL (i.e., 1000-1500 copies per tube) was claimed, but in our opinion, the high data scattering and poor temporal resolution of the published realtime plots do not allow LAMP performances to be accurately evaluated. 21 The analytical performances of the real-time electrochemical isothermal DNA amplifications so far developed appear promising, [13][14][15][16][17]21 but it is difficult to directly compare them, each being carried out under different conditions, using various DNA targets and redox probes.…”

An Integrated Real‐time Electrochemical LAMP Device for Pathogenic Bacteria Detection in Food

“…In our study, we used methylene blue (MB) as the DNA‐binding redox probe because it is efficiently intercalated into the DNA double helix by van der Waals, hydrogen bonding, and π‐π stacking interactions . The MB probe is far more suitable and attractive for use in real‐time electrochemical LAMP because of its strong ds‐DNA intercalating ability . The schematic principle for real‐time electrochemical LAMP for DNA amplification monitoring is described in Scheme .…”