Ratiometric Fluorescent Bioprobe for Highly Reproducible Detection of Telomerase in Bloody Urines of Bladder Cancer Patients

Abstract

Fluorescent bioprobes, as one of the most important tools, hold high promise for real-time analytical sensing of biological molecules and processes in live cells and organisms. Although several excellent bioprobes employ turn-on fluorescence intensity, such a sole responsive signal is readily perturbed by various experimental conditions. Herein, to improve the reproducibility and robustness, a ratiometric fluorescent bioprobe for telomerase activity detection has been developed. The ratiometric fluorescent bioprobe is designed on the use of two fluorescence dyes in parallel. One is red emissive aggregation-caused quenching (ACQ) dye, Cy5, as a control, molecularly labeled on the 5′-end of telomerase substrate oligonucleotides (TS primer). The other is water-soluble aggregation-induced emission (AIE) dye, Silole-R, as reporter of telomerase activity, nonemissive in buffer. In the presence of telomerase, the blue emission is enhanced by the added negatively charged sites for Silole-R to bind and aggregate, while the red emission is almost unchanged as stable internal reference. With the addition of incremental amounts of telomerase, the ratiometric emission intensity ratios (I 478/I 665) of this bioprobe gradually increase. Furthermore, the distinguishing of telomerase extracts from 20 bladder cancer bloody and 10 normal urine specimens confirms the practicality of this bioprobe. In contrast to previous turn-on bioprobes, these advanced experiments obtain higher reproducibility and positive result rate (100%) toward bladder cancer bloody urine specimens.

Fluorogenic Detection and Characterization of Proteins by Aggregation‐Induced Emission Methods

“…For real applications, fluorogenic detection without a reference is highly sensitive, but can be unreliable for quantification because a sole fluorogenic response, particularly AIE, is readily perturbed by other environmental factors. In this respect, by using a steady fluorophore, Cy5, at the TS primer, a ratiometric probe for telomerase activity detection was constructed that showed improved reproducibility and robustness, and was efficient for bloody urine samples from bladder cancer patients …”

“…In this respect, by using as teady fluorophore, Cy5, at the TS primer, ar atiometric probe for telomerase activity detection was constructed that showed improved reproducibility and robustness, and was efficient for bloody urine samples from bladder cancerpatients. [95] The quantification of enzymatic products can be carried out in af luorescencet urn-off format, particularly throught he disassemblyo fA IEgen-macromolecule complexes. This strategy works for degradation-type enzymes,such as protease ( Table 2, entries 33 and 34) and HAse (Table 2, entries 35 and 36).…”

Insight into the DNA adsorption on nitrogen-doped positive carbon dots

“…The management of the signal of the analytical methods through the ratiometric routine helps to eliminate the possible environment disturbance and improve the stability of the sensing performance. 20,34 Thus, the application and exhibition of the ratiometric strategy based on the system of uorescence quenching probe labeled DNA and N-CDs are the main advantages for such analytical methods. GO quenching uorescent probe also established a sensing platform to detect DNA but its detection performance with relatively low response (Fig.…”

An AIEgens and exonuclease III aided quadratic amplification assay for detecting and cellular imaging of telomerase activity

“…In our previous work, a series of telomerase activity detection protocols using AIE luminogens (AIEgens, weakly emissive when dissolved but strongly emissive when aggregated) have been reported [48][49][50][51][52][53][54]. Positive charged AIEgens showed weak fluorescence in solution.…”