Search

Protamine, a polycation, is biologically and medically relevant protein. Protamine exhibits a wide array of functions in biological processes like gene transfer, tissue and organogenesis, cell reproduction, etc. Medically, Protamine is the only clinically approved antidote for Heparin and is routinely used in various surgical interventions, and hence controlling Protamine dosing in patients is very crucial. Taking into account the medical significance of Protamine, designing simple, reliable and sensitive fluorescence sensors is highly desirable. In this work, we propose one such sensitive and reliable fluorescent sensor which is based on a template of dye-polyelectrolyte assembly constituting a molecular rotor dye, Thioflavin-T and an anionic synthetic polyelectrolyte, polystyrene sulfonate. The addition of Protamine, prompts drastic modulations in spectral features of dye-polyelectrolyte assembly which enables sensitive detection of Protamine in aqueous solution. Apart from sensitive detection, our sensing platform aids in highly selective sensing of Protamine compared to other proteins. Moreover, our sensor system is constructed on label-free, inexpensive, commercially available molecules posing as an advantage over other sensor systems which involve laborious synthesis protocols. Most importantly, our sensor template is able to sense Protamine in diluted serum sample, indicating the potential practical utility of our sensor system.

The inevitable importance of basic amino acids, arginine and lysine, in human health and metabolism demands construction of efficient sensor systems for them. However, there are only limited reports on the 'ratiometric' detection of basic amino acids which is further restricted by the use of chemically complex sensor molecules, which impedes their prospect for practical applications. Herein, we report a ratiometric sensor system build on simple mechanism of disassociation of novel emissive Thioflavin-T H-aggregates from heparin surface, when subjected to interaction with basic amino acids. The strong and selective electrostatic and hydrogen bonding interaction of basic amino acids with heparin leads to large alteration in photophysical attributes of heparin bound Thioflavin-T, which forms a highly sensitive sensor platform for detection of basic amino acids in aqueous solution. These selective interactions between basic amino acids and heparin allow our sensor system to discriminate arginine and lysine from other amino acids. This unique mechanism of dissociation of Thioflavin-T aggregates from heparin surface provides ratiometric response on both fluorimetric and colorimetric outputs for detection of arginine and lysine, and thus it holds a significant advantage over other developed sensor systems which are restricted to single wavelength detection. Apart from the sensitivity and selectivity, our system also provides the advantage of simplicity, dual mode of sensing, and more importantly, it employs an inexpensive commercially available probe molecule, which is a significant advantage over other developed sensor systems that uses tedious synthesis protocol for the employed probe in the detection scheme, an impediment for practical applications. Additionally, our sensor system also shows response in complex biological media of serum samples.

Discriminating proteins using simple methods is a highly ambitious task. Herein, we provide the first report of a cyclodextrin based supramolecular host-guest assembly, which without involving any specially designed receptor and transducer, yields a distinct fluorescence response i.e., turn-on response towards non-metalloproteins and turn-off response towards metalloproteins, and leads to their detection and discrimination.

Designing extrinsic fluorescence sensors for amyloid fibrils is a very active and important area of research. Recently, an ultrafast molecule rotor dye, Auramine O (AuO), has been projected as a fluorescent amyloid marker. It has been claimed that AuO scores better than the most extensively utilized gold-standard amyloid probe, Thioflavin-T (ThT). This advantage arises from the fact that AuO, in addition to its usual emission band (∼500 nm), also displays a large red-shifted emission band (∼560 nm), exclusively in the presence of human insulin fibril medium and not in the native protein or buffer media. On the contrary, for ThT, the emission maximum (∼490 nm) largely remains unchanged while going from protein to fibril. This otherwise unknown large red-shifted emission band of AuO, observed in the presence of human insulin fibrils, was tentatively attributed to a species formed upon fast proton dissociation from excited AuO. It was proposed that because of the long excited-state lifetime (∼1.8 ns) of AuO upon association with human insulin fibrils, this fast proton dissociation from excited AuO could be observed, which is otherwise not observed in buffer or native protein media, owing to its very short excited-state lifetime (∼1 ps). Herein, we show that despite the long excited-state lifetime of AuO in other fibrillar media (human serum albumin and lysozyme), the new red-shifted emission band at 560 nm is not observed, thus possibly suggesting a different origin of the red-shifted emission band of AuO in human insulin fibril medium. We convincingly show that this red-shifted band of AuO (∼560 nm) could be observed under conditions that promote dye aggregation, such as a premicellar concentration of surfactants and polyelectrolytes. These AuO aggregates display strong emission wavelength dependence of transient decay traces, similar to that for AuO in human insulin fibril medium. Detailed time-resolved emission spectral (TRES) measurements suggest that the AuO/premicellar surfactant and AuO/human insulin fibril system share similar features, such as a dynamic red-shift in TRES and an isoemissive point in the time-resolved area-normalized emission spectra, suggesting that the characteristic red-shifted emission band of AuO in human insulin fibril medium may arise from AuO aggregates.

Background: Amino acids are crucially involved in a myriad of biological processes. Any aberrant changes in physiological level of amino acids often manifest in common metabolic disorders, serious neurological conditions and cardiovascular diseases. Thus, devising methods for detection of trace amounts of amino acids becomes highly elemental to their efficient clinical diagnosis. Recently, the domain of developing optical sensors for detection of amino acids has witnessed significant activity which is the focus of the current review article. Methods: We undertook a detailed search of the peer-reviewed literature that primarily deals with optical sensors for amino acids and focuses on the use of different type of materials as a sensing platform. Results: Ninety-five papers have been included in the review, majority of which deal with optical sensors. We attempt to systematically classify these contributions based on the applications of various chemical and biological scaffolds such as polymers, supramolecular assemblies, nanoparticles, DNA, heparin etc for the sensing of amino acids. This review identifies that supramolecular assemblies and nanomaterial continue to be commonly used platforms to devise sensors for amino acids followed by surfactant assemblies. Conclusion: The broad implications of amino acids in human health and diagnosis have stirred a lot of interest to develop optimized optical detection systems for amino acids in recent years, using different materials based on chemical and biological scaffolds. We have also attempted to highlight the merits and demerits of some of the noteworthy sensor systems to instigate further efforts for constructing amino acids sensor based on unconventional concepts.

Recently, Auramine O (AuO) has been projected as a fluorescent fibril sensor, and it has been claimed that AuO has an advantage over the most extensively utilized fibril marker, Thioflavin-T (ThT), owing to the presence of an additional large red-shifted emission band for AuO, which was observed exclusively for AuO in the presence of fibrillar media and not in protein or buffer media. As fibrils are very rich in β-sheet structure, a fibril sensor should be more specific toward the β-sheet structure so as to produce a large contrast between the fibril form and native protein form, for efficient detection and in vitro mechanistic studies of fibrillation. However, in this report, we show that AuO interacts significantly with the native form of bovine serum albumin (BSA), which is an all-α-helical protein and lacks the β-sheet structure, which are the hallmarks of a fibrillar structure. This strong interaction of AuO with the native form of BSA leads to a large emission enhancement of AuO for the native protein itself, and leads to a low contrast between the BSA protein and its fibrils. More importantly, the large red-shifted emission band of AuO, reported in the presence of human insulin fibrils, and which was projected as its major advantage over ThT, is not observed in the presence of BSA fibrils as well as fibrils from other proteins, such as lysozyme, human serum albumin, and β-lactoglobulin. Thus, our results provide information on the universal applicability of the distinctive and claimed-to-be-advantageous photophysical features reported for AuO in human insulin fibrils towards fibrils from other proteins. Time-resolved fluorescence measurements also support the proposition of a strong interaction of AuO with native BSA. Additionally, tryptophan emission of the protein has been explored to further elucidate the binding mechanism of AuO with native BSA. Evaluation of thermodynamic parameters revealed that the binding of AuO with native BSA involved positive enthalpy and entropy changes, suggesting dominant contributions from hydrophobic and electrostatic interactions toward the association of AuO with native BSA. Molecular docking calculations have been performed to identify the principal binding location of AuO in native BSA.