Hasan Kurt

Dual-excitation upconverting nanoparticle and quantum dot aptasensor for multiplexed food pathogen detection.

In this report, a dual-excitation sensing method was developed using aptamer-functionalized quantum dots and upconverting nanoparticles, exhibiting Stokes and anti-Stokes type excitation profiles, respectively. Conjugation of the aptamer-functionalized luminescent nanoparticles with the magnetic beads, comprising short DNA sequences that were partially complementary to the aptamer sequences, enabled facile separation of the analyte-free conjugates for fluorescent measurement. UV-Visible spectroscopy, Circular Dichroism spectroscopy, Dynamic Light Scattering and Polyacrylamide Gel Electrophoresis techniques were used to characterize the aptamer probes developed. The target-specific luminescent conjugates were applied for multiplex detection of model food pathogens, Salmonella typhimurium, and Staphylococcus aureus, in which the fluorescent emission spectra were obtained under UV excitation at 325nm for quantum dots and NIR excitation at 980nm for upconverting nanoparticles, respectively. The dual-excitation strategy was aimed to minimize cross-talk between the luminescent signals for multiplexed detection, and yielded limit of detection values of 16 and 28cfumL(-1) for Staphylococcus aureus, and Salmonella typhimurium, respectively. By employing a greater number of quantum dots and upconverting nanoparticles with non-overlapping fluorescent emissions, the proposed methodology might be exploited further to detect several analytes, simultaneously.

Employment of nanomaterials in polymerase chain reaction: insight into the impacts and putative operating mechanisms of nano-additives in PCR

The unique ability to rapidly amplify low copy number DNA has made in vitro Polymerase Chain Reaction one of the most fundamental techniques in modern biology. In order to harness this technique to its full potential, certain obstacles such as nonspecific by-products, low yield and complexity of GC rich and long genomic DNA amplification need to be surmounted. As in vitro PCR does not have any regulatory mechanisms unlike its counterpart in vivo DNA replication machinery, scientists often use a number of additives like glycerol, betaine, dimethyl sulphoxide and formamide in order to achieve the perfection of in vivo systems. In the last two decades nanotechnology has provided excellent solutions to many classical problems in various scientific fields including biotechnology and recently the PCR technique has begun to benefit from this so called “Nano Era”. In this review, the impacts of several nanomaterials on PCR efficiency, specificity and fidelity are described in accordance with the recent literature. Putative interaction mechanisms between nanomaterials and primary PCR components are also addressed in a comprehensive manner.

How to make nanobiosensors: surface modification and characterisation of nanomaterials for biosensing applications

This report aims to provide the audience with a guideline for construction and characterisation of nanobiosensors that are based on widely used affinity probes including antibodies and aptamers and nanomaterials such as carbon-based nanomaterials, plasmonic nanomaterials and luminescent nanomaterials. The affinity probes and major methodologies that have been extensively used to make nanobiosensors, such as thiol–metal interactions, avidin–biotin interaction, π-interactions and EDC–NHS chemistry, were described with the most recent examples from the literature. Characterisation techniques that have been practised to validate nanoparticle surface modification with antibodies and aptamers, including gel electrophoresis, ultraviolet-visible spectrophotometry, dynamic light scattering and circular dichroism were described with examples. This report mainly covers the reports published between 2014 and 2017.

Conformation-mediated Förster resonance energy transfer (FRET) in blue-emitting polyvinylpyrrolidone (PVP)-passivated zinc oxide (ZnO) nanoparticles

Homopolymers, such as polyvinylpyrrolidone (PVP), are commonly used to passivate the surface of blue-light emitting ZnO nanoparticles during colloid nucleation and growth. However, although PVP is known to auto-fluoresce at 400nm, which is near the absorption edge of ZnO, the impact of PVP adsorption characteristics on the surface of ZnO and the surface-related photophysics of PVP-capped ZnO nanoparticles is not well understood. To investigate, we have synthesized ZnO nanoparticles in solvents containing PVP of 3 concentrations-0.5, 0.7, and 0.11gmL-1. Using time-domain NMR, we show that the adsorbed polymer conformation differs with polymer concentration-head-to-tail under low concentration (e.g., 0.05gmL-1) and looping, then train-like, with increasing concentration (e.g., 0.07gmL-1 and 0.11gmL-1, respectively). When the surface-adsorbed PVP is entrained, the surface states of ZnO are passivated and radiative emission from surface trap states is suppressed, allowing emission to be dominated by exciton transitions in the UV (ca. 310nm). Moreover, the reduced proximity between the PVP molecule and the ZnO gives rise to increased efficiency of energy transfer between the exciton emission of ZnO and the HOMO-LUMO absorption of PVP (ca. 400nm). As a result, light emission in the blue is enhanced in the PVP-capped ZnO nanoparticles. We thus show that the emission properties of ZnO can be tuned by controlling the adsorbed PVP conformation on the ZnO surface via the PVP concentration in the ZnO precipitation medium.

Characterization of a dual biotin tag for improved single stranded dna production

Generation of single-stranded DNA plays a key role in many biotechnology applications including production of aptamers, single strand conformation polymorphism, nuclease S1 mapping, pyrosequencing, genosensors, probe preparation and labelling, subtractive hybridization as well as nucleic acid sensing and microarrays. Several methods are available in the literature to produce single-stranded DNA from double-stranded DNA templates, such as extraction of the sense strand from denaturing gels, asymmetric PCR, use of streptavidin–biotin interaction, and some alternative methods, including enzymatic digestion of the negative strand by either lambda exonuclease or T7 Gene 6 exonuclease. In this report, a detailed characterization of a dual biotin tag method to generate single-stranded DNA from the random oligonucleotide library is presented. Unlike the traditional streptavidin–biotin method that uses single biotin tagged molecules during separation, this novel technique is based on a dual biotin molecule covalently attached to the 5′ end of the negative strand. The improved technique takes less than one hour as a consequence of the eliminated alkali treatment step, which makes this procedure the shortest procedure described in the literature so far for single-stranded DNA production. The method can achieve a single-stranded DNA yield around 75% from the corresponding DNA template in Tris–HCl buffer. A number of parameters, such as the effect of different elution buffers and heat treatments, spontaneous release of streptavidin from the magnetic bead surface, loss of beads during consecutive washes, aggregation of the beads, were investigated to reveal the optimal conditions for single-stranded DNA production. FTIR, DLS, SEM, and electrophoresis techniques were used for characterization studies.