Chemiluminescent Protease Probe for Rapid, Sensitive, and Inexpensive Detection of Live Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a top-ten cause of death worldwide. Successful treatment is often limited by insufficient diagnostic capabilities, especially at the point of care in low-resource settings. The ideal diagnostic must be fast, be cheap, and require minimal clinical resources while providing high sensitivity, selectivity, and the ability to differentiate live from dead bacteria. We describe here the development of a fast, luminescent, and affordable sensor of Hip1 (FLASH) for detecting and monitoring drug susceptibility of Mycobacterium tuberculosis ( Mtb ). FLASH is a selective chemiluminescent substrate for the Mtb protease Hip1 that, when processed, produces visible light that can be measured with a high signal-to-noise ratio using inexpensive sensors. FLASH is sensitive to fmol of recombinant Hip1 enzyme in vitro and can detect as few as thousands of Mtb cells in culture or in human sputum samples within minutes. The probe is highly selective for Mtb compared to other nontuberculous mycobacteria and can distinguish live from dead cells. Importantly, FLASH can be used to measure antibiotic killing of Mtb in culture with greatly accelerated timelines compared to traditional protocols. Overall, FLASH has the potential to enhance both TB diagnostics and drug resistance monitoring in resource-limited settings.

Modular Access to Diverse Chemiluminescent Dioxetane‐Luminophores through Convergent Synthesis

“…Numerous groups over the world have already taken advantage of this discovery to prepare various chemiluminescent probes, which are based on these new luminophores. [9][10][11][12][13][14][15][16] After more than 30 years of dormancy in this research area, we have succeeded in taking a major step forward, by understanding how to improve and control light emission properties under aqueous conditions. Chemiluminescence applications of dioxetane luminophores is now poised for broader use in a variety of exciting contexts.…”

“…[17] Implementation of this synthetic route by our group and others has enabled the preparation of numerous adamantyl-phenoxy-dioxetane derivatives, although this pathway requires multi-step synthesis, and harsh reaction conditions, such as; the need for a strong base in the Wittig-Horner step. [9,12,[18][19][20][21][22][23][24] In addition, the scope of available meta phenoxy-aldehydes as starting materials is also limited. Due to the aforementioned limitations, a more robust and efficient synthesis of the enolether intermediate should be developed to enable the preparation of new challenging dioxetane luminophores.…”

“…This discovery has enabled, for the first time, the use of dioxetane chemiluminescent luminophores as single component probes for various applications that require physiological conditions. Numerous groups over the world have already taken advantage of this discovery to prepare various chemiluminescent probes, which are based on these new luminophores [9–16] . After more than 30 years of dormancy in this research area, we have succeeded in taking a major step forward, by understanding how to improve and control light emission properties under aqueous conditions.…”

“…In 2000, the group of Sammes reported a five‐step synthesis that utilized a key Wittig–Horner cross‐coupling reaction to obtain the enolether precursor (Figure 2B) [17] . Implementation of this synthetic route by our group and others has enabled the preparation of numerous adamantyl‐phenoxy‐dioxetane derivatives, although this pathway requires multi‐step synthesis, and harsh reaction conditions, such as; the need for a strong base in the Wittig–Horner step [9, 12, 18–24] . In addition, the scope of available meta phenoxy‐aldehydes as starting materials is also limited.…”

Synthesis and Evaluation of Ubiquitin–Dioxetane Conjugate as a Chemiluminescent Probe for Monitoring Deubiquitinase Activity

“… 29 , 30 Such probes produce extremely low background signal, due to their high stability toward spontaneous hydrolysis. 31 The enzymatic responsive group used to mask the phenoxy-dioxetane luminophores were all composed of small molecules or short peptides. 32 − 34 A responsive group based on a full protein substrate, attached to the dioxetane luminophore, has never been demonstrated.…”