Sohail Anjum Shahzad

Controlled self-assembly of small molecule probes and the related applications in bioanalysis

Fluorescence spectroscopy is widely used in basic research, disease diagnosis, environmental monitoring, and the development of novel bioanalytical techniques. We mainly focus on the changes in fluorescence signal originated from the controlled self-assembly of small molecule probes, including aggregation caused quenching, aggregation induced emission, controlled turn-on of probe monomer emission, and the tunable monomer-excimer transition. Recent developments in the related bioanalytical techniques have been reviewed.

A fluorescence turn-on detection of copper(II) based on the template-dependent click ligation of oligonucleotides

In this work, a fluorescence turn-on method for copper(II) detection is reported. A molecular beacon (MB) was designed as a template. Cu(2+) was reduced to Cu(+) in the presence of a reductant (ascorbic acid). Two short single-stranded oligonucleotides one was labeled with a 5-alkyne and the other with 3-azide group, proceeded a template-dependent chemical ligation through the Cu(I)-catalyzed azide-alkyne cycloaddition. The newly generated click-ligated long chain oligonucleotide, which was complementary to the MB, opened the MB hairpin structure and resulted in a turn on fluorescence. The increase in fluorescence intensity is directly proportional to the amount of Cu(2+) added to the assay solution. The present assay is quite sensitive and allows the detection of 2 nM Cu(2+). The described assay also exhibits high selectivity over other metal ions.

Fluorescence turn-on detection of alkaline phosphatase activity based on controlled release of PEI-capped Cu nanoclusters from MnO2 nanosheets

A fluorescence turn-on assay for alkaline phosphatase (ALP) activity is developed through the controlled release of polyethyleneimine-capped copper nanoclusters (PEI-capped CuNCs) from the MnO2 nanosheets. In an aqueous solution, the positively charged PEI-capped CuNCs could be adsorbed onto the surface of the negatively charged MnO2 nanosheets. Such adsorption through favorable electrostatic interactions could efficiently quench the nanocluster fluorescence emission via resonance energy transfer from the PEI-capped CuNCs to the MnO2 nanosheets. 2-Phospho-l-ascorbic acid (AAP) could be hydrolyzed to l-ascorbic acid (AA) in the presence of ALP. AA could reduce MnO2 into Mn2+ and trigger the disintegration of the MnO2 nanosheets. As a result, the CuNCs were released and the quenched fluorescence was recovered efficiently. The detection strategy is simple, inexpensive, sensitive, selective, with low toxicity, and has better biocompatibility. The newly fabricated biosensor for ALP activity will potentially make it a robust candidate for numerous biological and biomedical applications.

Nucleic acid-controlled quantum dots aggregation: A label-free fluorescence turn-on strategy for alkaline phosphatase detection

Based on the controlled aggregation of quantum dots (QDs), a valid, reliable, and label-free fluorescence turn-on strategy is established for the detection of alkaline phosphatase activity. The aqueous solution of anionic QDs exhibits intense fluorescence. However, the addition of cationic polymer (poly-1) significantly quenched the fluorescence of the QDs via their induced aggregation. While short 3-phosphorylated DNA (DNA-P) could not be extended by terminal deoxynucleotidyl transferase (TdT) and therefore, fluorescence of the QDs was recovered negligibly. The effective elimination of phosphate group of DNA-P in the presence of alkaline phosphatase (ALP) produced 3-OH termini and the resulting DNA could be sufficiently extended by TdT. The presence of greater binding strength between the elongated DNA and poly-1 is very crucial to compete with the poly-1/QDs aggregates and release the QDs. Turned-on fluorescence emission is observed due to the efficient release of the QDs. A novel strategy for alkaline phosphatase detection is therefore established. Our method is quite sensitive and selective, as low as 0.1 mU/mL ALP can be easily detected.

Synthesis of silica nanoparticles doped with [Ru(bpy) 3 ] 2+ and decorated with silver nanoclusters for the ratiometric photoluminescent determination and intracellular imaging of Cu(II) ions

AbstractA sensitive and selective luminescent nanoprobe (referred to as DEPN) is designed for the determination of Cu(II) ions. DEPN shows two emission peaks, one at 602xa0nm and caused by [Ru(bpy)3]2+, the other (peaking at 500xa0nm) caused by silver nanoclusters (AgNCs). The luminescence of the ruthenium complex is inert and acts as the internal reference signal, while that of the AgNCs is quenched by Cu(II) ions. Under 350xa0nm photoexcitation, a linear relationship is found between the ratio of emission intensity (I500/I602) and the concentration of Cu(II) ions in the 0.1–16xa0μM range. The detection limit of Cu(II) ions is estimated to be 50xa0nM. The DEPN was applied to the determination of Cu(II) ions in (spiked) water samples, and to intracellular imaging of Cu(II) in HeLa cells using confocal fluorescence microscopy.n Graphical abstractSchematic presentation of a dual-emission photoluminescence nanoprobe for selective Cu2+ sensing. The emission of the Ag nanoclusters (at 500xa0nm) is quenched by Cu2+, while that of the Ru(bpy)32+-doped silica nanoparticles (at 602xa0nm) remains unchanged and serves as a reference.

A real-time fluorescence turn-on assay for acetylcholinesterase activity based on the controlled release of a perylene probe from MnO2 nanosheets

We exploit a real-time perylene probe fluorescence turn-on method to detect acetylcholinesterase (AChE) activity through the selective decomposition of MnO2 nanosheets. The surface of the MnO2 nanosheets is negatively charged. The perylene probe (P-4C+) has four positively charged quaternary ammonium groups. When mixed together, P-4C+ attached to the surface of the MnO2 nanosheets through electrostatic attractive interactions. The fluorescence of P-4C+ was effectively quenched by the MnO2 nanosheets. Acetylthiocholine (ATCh) could be hydrolyzed to thiocholine by AChE. Thiocholine is a reducing agent. It could reduce MnO2 nanosheets to Mn2+ and thus trigger the decomposition of the MnO2 nanosheets. As a result, P-4C+ was released and the fluorescence of P-4C+ was restored. The AChE inhibitor restrained the catalytic activity of AChE and resulted in a reduced fluorescence recovery of P-4C+. Our assay method is simple and selective, with low toxicity and better biocompatibility, which would facilitate biological and biomedical applications associated with AChE activity.

Silver nanoclusters capped silica nanoparticles as a ratiometric photoluminescence nanosensor for the selective detection of I− and S2−

A novel and efficient approach has been established for the synthesis of silver nanoclusters capped silica nanoparticles (SiO2@AgNCs). These nanoclusters (AgNCs) capped silica nanoparticles were utilized as a novel ratiometric photoluminescence (PL) nanosensor for extremely sensitive and selective detection of I- and S2- ions. The AgNCs were prepared in situ on the silica nanoparticles through polyethyleneimine (PEI) template approach. While dual PL emissions of AgNCs (at 500xa0nm) and luminescent silica nanoparticles (at 602xa0nm) formed the basis for the ratiometric sensing. The PL emission of AgNCs was strongly quenched by I- (or S2-), while that of luminescent silica nanoparticles was hardly affected. The PL emission intensity ratio of AgNCs and the luminescent silica nanoparticles was defined as I500/I602. A good linear relationship between the I500/I602 value and the concentration of I- (or S2-) was observed, and the limit of detection (LOD) was estimated to be 57xa0nM for I- and 62xa0nM for S2-. In addition, the fluorescence images of the SiO2@AgNCs nanosensor changed from white to orange upon exposure to different concentrations of I- (or S2-) (0-250xa0μM), which could be clearly distinguished by the naked eye. The SiO2@AgNCs nanosensor exhibited good selectivity against other analytes, and I- or S2- ions could be separately detected via the introduction of proper masking agents. Furthermore, the detection of I- and S2- in real water samples was also demonstrated.

Tuning of the perylene probe excimer emission with silver nanoparticles

Silver nanoparticles (Ag NPs) enhanced perylene probe excimer emission is reported for the first time. It was observed that strong interactions between the perylene probe and the Ag NPs induced co-aggregation. As a result, a new in situ generated plasmonic absportion band of the Ag NPs at longer wavelength emerged. The monomer emission of the perylene probe was efficiently quenched, and dramatically enhanced probe excimer emission was observed. A remarkable emission enhancement of over 1000 fold was obtained compared to anionic polymers and other nanoparticles. The excimer emission intensity could be finely modulated by the size of the Ag NPs and the functionalities of the perylene probe. The observed Ag NPs enhanced perylene probe excimer emission shows good potential for the development of novel sensing techniques for various bioanalytical applications.

A fluorescence turn on assay for alkaline phosphatase based on the Cu2+ catalyzed Fenton-like reaction

A fluorescence turn-on assay was established for ALP (alkaline phosphatase) based on Cu(2+) catalyzed Fenton-like reaction and Graphene Oxide (GO). GO was utilized to quench the fluorescence of fluorescein (FAM) labeled single strand DNA (F-DNA). ALP can remove the phosphate group in sodium ascorbyl phosphate (SAP), and convert it into reducing ascorbate. Highly reactive hydroxyl radicals (·OH) were generated in the presence of ascorbate and Cu(2+) through the Fenton-like reaction. The reactive radicals generated in situ caused the cleavage of F-DNA into small fragments. When GO was added, the fluorescence emission of the sample without ALP was quenched and fluorescence emission recovered in the presence of ALP. The intensity of the recovered fluorescence was directly related to the concentration of ALP in the assay solution, and a sensitive and selective facile ALP assay is therefore established.

Benzo[ghi]perylene and coronene: ratiometric fluorescent probes for the sensing of microenvironment changes and micelle formation in aqueous medium

Benzo[ghi]perylene and coronene were used as ratiometric fluorescent probes to monitor microenvironment changes and monomer-micelle transition for the first time. The perturbations in vibronic bands in the emission spectra of probes displayed microenvironment sensing behavior in various solvents and surfactants. The monomer–micelle transitions of anionic, cationic and neutral surfactants were monitored by changes in the ratios of vibronic band intensities. Clear CMC values were obtained, which is an advantage over the conventional probes such as pyrene with clear CMC values sometimes difficult to obtain due to less steep changes of S-shaped plots. They revealed a redshift in emission which offers a good substitute in situations where the fluorescence of pyrene and the background fluorescence of the assay mixture interfere with each other. BzP exhibited reverse behavior in the perturbations of vibronic band intensities in anionic SDS and SLS relative to cationic and non-ionic surfactants, which was not observed when pyrene was used. In addition, the vibronic band ratios of BzP are thermally more stable than those of pyrene, which is advantageous since strict assay temperature control may not be necessary. These probes are thus helpful for various applications in the areas of sensing and interface science.