A competitive and reversible deactivation approach to catalysis-based quantitative assays

Abstract

Catalysis-based signal amplification makes optical assays highly sensitive and widely useful in chemical and biochemical research. However, assays must be fine-tuned to avoid signal saturation, substrate depletion and nonlinear performance. Furthermore, once stopped, such assays cannot be restarted, limiting the dynamic range to two orders of magnitude with respect to analyte concentrations. In addition, abundant analytes are difficult to quantify under catalytic conditions due to rapid signal saturation. Herein, we report an approach in which a catalytic reaction competes with a concomitant inactivation of the catalyst or consumption of a reagent required for signal generation. As such, signal generation proceeds for a limited time, then autonomously and reversibly stalls. In two catalysis-based assays, we demonstrate restarting autonomously stalled reactions, enabling accurate measurement over five orders of magnitude, including analyte levels above substrate concentration. This indicates that the dynamic range of catalysis-based assays can be significantly broadened through competitive and reversible deactivation.

Direct fluorogenic detection of palladium and platinum organometallic complexes with proteins and nucleic acids in polyacrylamide gels

“…The inefficiency of Hyd to activate the probe in presence of Pt compounds could be explained by the inability of Hyd to reduce Pt 2+ into Pt 0 . These results are exciting because only a few fluorescent sensors, such as rhodamine triazole 23 , diethyldithiocarbamate-rhodamine B 24 , resorufin allyl ether 8 , and Pittsburgh green allyl ether 10 are sensitive to Pt compounds. All published probes respond differently to various Pt compounds; however, the sensitivity of only a few of them has been tested against anticancer complexes.…”

“…Previously produced probes based on Pittsburgh Green 9 , 10 , dichlorofluorescein 16 , resorufin 8 , 4-hydroxynaphthalimide 17 are sensitive and allow facile detection of Pd in quantitative assays. However, they frequently retain a relatively high level of fluorescence under UV making them excellent probes for spectrophotometric assays and microscopy but less effective for the use with an UV transilluminator.…”

“…Majority of chemical sensors for the detection of noble metals utilizes their catalytic properties 6 with Pd sensors often based on allyl or propargyl carbamates, carbonates, or ethers of phenolic fluorophores [6][7][8][9][10] . The fluorescent activation of the probes is based on the Tsuji-Trost allylic oxidative insertion mechanism 11 .…”

High throughput analysis enables high throughput experimentation in pharmaceutical process research

“…[65][66][67] While effective, these analytical techniques were very expensive and somewhat cumbersome. We worked closely with Professor Kaz Koide at University of Pittsburgh to develop a series of highly sensitive catalysis-based fluorogenic 68 and chromogenic 69,70 reagents and assay protocols for 'point of use' assessment of the concentration of palladium, our most frequently encountered metal impurity.…”

“…Direct MS measurements using DESI or DART ionization are increasingly being used, 109 and we recently achieved the high throughput analysis of 1536 well microplates of reaction mixtures using MALDI MS in 8 minutes (3 samples per second), 110 which compares favorably with the speed of droplet microfluidic MS analysis reported by Kennedy 111,112 and others. In addition, colorimetric, fluorimetric and CD-based sensors such as those developed by Anslyn, 113 Wolf 114 Joyce 115,116 and Koide [68][69][70] are increasingly being used in high throughput analysis of reaction mixtures. 117…”