Media Dependent Switching of Selectivity and Continuous near Infrared Turn-on Fluorescence Response through Cascade Interactions from Noncovalent to Covalent Binding for Detection of Serum Albumin in Living Cells

Abstract

Abnormal level of proteins is proved to be associated with diseases. Thus, protein sensing is helpful for clinical diagnosis and therapy. However, there is a great variety of protein species and relatively low concentration of each protein in complicated biological systems including other nonprotein biomolecules. Therefore, it remains challenging to develop an effective method for detecting protein with high selectivity and sensitivity. Herein, a new self-assembly method based on a robust dye SQSS of which two squaraine molecules were conjugated through disulfide bond was developed for highly selective and sensitive detection of serum albumin (SA) in aqueous solution and live cells. SQSS can self-assemble into "compact" aggregates, offering "inert" disulfide group and very low background fluorescence through the combination of aggregation quenching and homogeneous fluorescence resonance energy transfer (homoFRET) quenching. The response of SQSS to SA undergoes two cascade stages. At the first stage, SA drives the compact assemblies of SQSS to form loose ones with fast speed (30 s) through noncovalent interaction, resulting in the enhancement of fluorescence to some extent. In this loose assembly state, the disulfide bond in SQSS is reactive. At the second stage, the Cys34 in SA slowly induced further disassembly through covalent binding with reactive disulfide bond, resulting in fluorescence further increasing and SQSS labeling to SA that cannot be displaced by site binding ligands of SA. The self-assemblies of SQSS can selectively detect SA with continuous near-infrared (NIR) turn-on fluorescence response in 100% aqueous buffer solution. In addition, SQSS showed the potential application of imaging SA in living cells. On the other hand, the loose assembly state of SQSS was also achieved in aqueous solution with 20% CH 3 CN. In this media, thiol-containing glutathione (GSH) caused the disassembly of SQSS with turn-on fluorescence response through interaction with disulfide bond. SQSS can selectively recognize GSH over other amino acids even in the presence of other sulfhydryl amino acids. As a proof-of-concept method, the molecular self-assembly through multisteps interactions would provide an ideal strategy for detection and live-cell imaging of biorelated molecules with high selectivity and signal-to-noise ratio.

New “Destruction Seek to Survive” Strategy Based on a Serum Albumin Assembly with a Squaraine Molecule for the Detection of Peroxynitrite

“…To complement the above studies, a competition assay was performed with two known binders dansylamide (DNSA, for site I) and dansylproline (DP for site II) in Figure S11. 47 Because SQDC only partially enters the cavity of BSA, fewer binders are required in the competition experiment. After adding ONOO − , the SQ segment in the assembly can better enter the cavity due to the "destruction seek to survive" strategy, and increase the required competitive dose.…”

“…The results demonstrated that the reaction of SQDC@BSA with ONOO – afforded a large fluorescence change, thus amplifying the fluorescence change. To complement the above studies, a competition assay was performed with two known binders dansylamide (DNSA, for site I) and dansylproline (DP for site II) in Figure S11 . Because SQDC only partially enters the cavity of BSA, fewer binders are required in the competition experiment.…”

Serum Albumin Inspired Self-Assembly/Disassembly of a Fluorogenic Nanoprobe for Real-Time Monitoring and Quantification of Urinary Albumin with Live Cell Imaging Application

“…The probe HJRA (2 μM) displays fluorescent-silent emission band at 598 nm (Figure S9) in ∼100% PBS buffer (pH 7.4, containing 0.2% DMSO) upon excitation at 539 nm. The nonemissive status may be due to the formation of loosely packed nanoaggregates resulting aggregation caused quenching (ACQ) effect − and the presence of twisted intramolecular charge transfer process. Therefore, to establish the involvement of TICT process, change in photophysical (UV–vis absorption and fluorescence) behavior of probe was explored.…”

“…Besides, there are also few reports where some near-infrared (NIR) dyes bind with HSA to generate HSA–dye nanocomposites for applications in bioimaging, tumor therapeutic diagnosis, and potential drug delivery . However, Thayumanavan and colleagues have successfully employed the polymer–nanoparticle - disassembly strategy as a unique technique for the detection of both protein and enzyme. − On the contrary, only a few examples exist where this disassembly technique was utilized successfully for small-molecule derived self-assembled nanoaggregated probes for the HSA and BSA detection. − …”

Solo Smart Fluorogenic Probe for Potential Cancer Diagnosis and Tracking in Vivo Tumorous Lymphatic Systems via Distinct Emission Signals

“…Although many efficient fluorescent probes have been developed over the years for the specific detection of the albumin proteins (HSA/BSA) based on the probe-albumin chelation, many of these probes suffer from limitations, in terms of selectivity, high background signal, excitation/emission in the lower wavelength regions, and small Stokes shift, which essentially restricted their possible exploration in the in vivo biological models for advanced clinical investigations. − On the other hand, NIR dyes such as Indocyanine Green (ICG), Cy5.5, and IRDye800CW have been used for detecting the location of SLNs in various tumors . However, these dyes involve a small Stokes shift (interference from excitation light) and the lack of any associated turn-on response mechanism.…”