Chittanon Buranachai

A FRET based aptasensor coupled with non-enzymatic signal amplification for mercury (II) ion detection

In this work, the idea of incorporating a non-enzymatic signal amplification with a regular aptasensor was tested. In this proof of principle, the sensor was designed for the detection of mercury (II) ions (Hg(2+)) based on the Förster Resonance Energy Transfer (FRET), and the catalyzed hairpin assembly (CHA) technique that was used as the signal amplification method. This sensor comprised a mercury aptamer-catalyst complex (Apt-C) and two types of hairpin DNA: H1 labeled with fluorescein and H2 labeled with tetramethylrhodamine. In the presence of Hg(2+), two facing thymine bases in the mercury aptamer strand were coordinated with one mercury ion. This caused the release of the catalyst for the catalyzed hairpin assembly (CHA) reaction that turned H1 and H2 hairpins into H1-H2 hybrids. FRET was then used to report the hairpin-duplex transformation. The sensor showed excellent specificity towards Hg(2+) over other possible interfering cations present at even a 100 fold greater concentrations. It had a linear range of 10.0-200.0nM, and a good detection limit of 7.03±0.18nM, which is lower than the regulatory mercury limit for drinking water (10nM or 2ppb). The sensor was used to detect spiked Hg(2+) in nine real surface water samples collected from three different areas. Acceptable recoveries and small standard deviations indicated that the sensor was practically applicable, and the proposed idea to incorporate a CHA amplification in a regular aptasensor was not only feasible but beneficial. The same principles can be applied to develop sensors for various different targets.

A Novel Reconfigurable Optical Biosensor Based on DNA Aptamers and a DNA Molecular Beacon

In order to alter a typical molecular aptamer beacon (MAB) to detect a different analyte there is currently a need to change the whole sensor unit including the expensive labeling fluorophores. In this work a DNA-based reconfigurable molecular aptamer beacon was developed. It is composed of two parts: a variable part and a constant part. The variable part comprises an aptamer strand and its complementary strand while the constant part is an oligonucleotide doubly labeled with a Förster Resonance Energy Transfer (FRET) pair and the two parts become joined via DNA hybridization. The sensor exists in two conformations: a folded (high FRET) and an unfolded (low FRET) in the absence and presence of the aptamer-target binding respectively. This sensor can be reconfigured by washing away the aptamer and the complementary strand using proper complementary strands, called washers. As a proof of the principle, a sensor that bound the enzyme thrombin, an analyte with a strong binding, was first constructed and then reconfigured to bind adenosine, selected as an analyte with a weak binding. We believe that the design is of universal use applicable to many types of aptamers.

Enhancing capacitive DNA biosensor performance by target overhang with application on screening test of HLA-B*58:01 and HLA-B*57:01 genes

A highly sensitive label-free DNA biosensor based on PNA probes immobilized on a gold electrode was used to detect a hybridization event. The effect of a target DNA overhang on the hybridization efficiency was shown to enhance the detected signal and allowed detection at a very low concentration. The sensors performances were investigated with a complementary target that had the same length as the probe, and the signal was compared to the target DNAs with different lengths and overhangs. A longer target DNA overhang was found to provide a better response. When the overhang was on the electrode side the signal enhancement was greater than when the overhang was on the solution side due to the increased thickness of the sensing surface, hence produced a larger capacitance change. Using conformationally constrained acpcPNA probes, double stranded DNA was detected sensitively and specifically without any denaturing step. When two acpcPNA probes were applied for the screening test for the double stranded HLA-B*58:01 and HLA-B*57:01 genes that are highly similar, the method differentiated the two genes in all samples. Both purified and unpurified PCR products gave comparable results. This method would be potentially useful as a rapid screening test without the need for purification and denaturation of the PCR products.

Excited state free energy calculations of Cy3 in different environments

Cy3, a cyanine dye, is one of the most widely used dyes in investigating the structure and dynamics of biomolecules by means of fluorescence methods. However, Cy3 fluorescence emission is strongly competed by trans-cis isomerization, whose efficiency is dictated by the isomerization energy barrier and the environment of Cy3. The fluorescence quantum yield of Cy3 is very low when the dye is free in homogeneous solution but it is considerably enhanced in an environment that rigidifies the structure, e.g. when it is attached to a DNA strand. In this work, the barriers for isomerization on the excited state of free Cy3, and Cy3 attached to single- and double-stranded DNA in methanol, are presented. The free energy and subsequently the isomerization barrier calculations are performed using the umbrella sampling technique with the weighted histogram analysis method. The hybrid quantum mechanics/molecular mechanics (QM/MM) approach is employed to provide the potential energy surfaces for the excited state dynamics simulations in umbrella sampling. The semiempirical floating occupation molecular orbital configuration interaction method is used for electronic excited state calculations of the QM region (Cy3). From the free energy calculations, the barrier of Cy3 attached to the single-stranded DNA is highest, in agreement with previously reported experimental results. This is likely due to the stacking interaction between Cy3 and DNA. Such a stacking interaction is likely associated with steric hindrance that prevents the rotation around the conjugated bonds of Cy3. If Cy3 experiences high steric hindrance, it has a higher isomerization barrier and thus the efficiency of fluorescence emission increases.