Abootaleb Sedighi

A proposed mechanism of the influence of gold nanoparticles on DNA hybridization.

A combination of gold nanoparticles (AuNPs) and nucleic acids has been used in biosensing applications. However, there is a poor fundamental understanding of how gold nanoparticle surfaces influence the DNA hybridization process. Here, we measured the rate constants of the hybridization and dehybridization of DNA on gold nanoparticle surfaces to enable the determination of activation parameters using transition state theory. We show that the target bases need to be detached from the gold nanoparticle surfaces before zipping. This causes a shift of the rate-limiting step of hybridization to the mismatch-sensitive zipping step. Furthermore, our results propose that the binding of gold nanoparticles to the single-stranded DNA segments (commonly known as bubbles) in the duplex DNA stabilizes the bubbles and accelerates the dehybridization process. We employ the proposed mechanism of DNA hybridization/dehybridization to explain the ability of 5 nm diameter gold nanoparticles to help discriminate between single base-pair mismatched DNA molecules when performed in a NanoBioArray chip. The mechanistic insight into the DNA-gold nanoparticle hybridization/dehybridization process should lead to the development of new biosensors.

Kras gene codon 12 mutation detection enabled by gold nanoparticles conducted in a nanobioarray chip.

This study employs a nanobioarray (NBA) chip for multiple biodetection of single base pair mutations at the Kras gene codon 12. To distinguish between the mutant and wild-type target DNAs, current bioarray methods use high-temperature hybridization of the targets to the allele-specific probes. However, these techniques need prior temperature optimization and become harder to implement in the case of the detection of multiple mutations. We aimed to detect these mutations at a single temperature (room temperature), enabled by the use of gold nanoparticles (AuNPs) on the bioarray created within nanofluidic channels. In this method, a low amount of target oligonucleotides (5fmol) and polymerase chain reaction (PCR) products (300pg) were first loaded on the AuNP surface, and then these AuNP-bound targets were introduced into the channels of a polydimethylsiloxane (PDMS) glass chip. The targets hybridized to their complementary probes at the intersection of the target channels to the pre-printed oligonucleotide probe lines on the glass surface, creating a bioarray. Using this technique, fast and high-throughput multiple discrimination of the Kras gene codon 12 were achieved at room temperature using the NBA chip, and the specificity of the method was proved to be as high as that with the temperature stringency method.

DNA Microarray-Based Diagnostics

The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications.

Dip-in Indicators for Visual Differentiation of Fuel Mixtures Based on Wettability of Fluoroalkylchlorosilane-Coated Inverse Opal Films

We have developed the dip-in indicator based on the inverse opal film (IOF) for visual differentiation of organic liquid mixtures, such as oil/gasoline or ethanol/gasoline fuel mixtures. The IOF consists of a three-dimensional porous structure with a highly ordered periodic arrangement of nanopores. The specularly reflected light at the interface of the nanopores and silica walls contributes to the structural color of the IOF film. This color disappears when the nanopores are infiltrated by a liquid with a similar refractive index to silica. The disappearance of the structural color provides a means to differentiate various liquid fuel mixtures based on their wettability of the nanopores in the IOF-based indicators. For differentiation of various liquid mixtures, we tune the wettability threshold of the indicator in such a way that it is wetted (color disappears) by one liquid but is not wetted by the other (color remains). Although colorimetric differentiation of liquids based on IOF wettability has been reported, differentiation of highly similar liquid mixtures require complicated readout approaches. It is known that the IOF wettability is controlled by multiple surface properties (e.g., oleophobicity) and structural properties (e.g., neck angle and film thickness) of the nanostructure. Therefore, we aim to exploit the combined tuning of these properties for differentiation of fuel mixtures with close compositions. In this study, we have demonstrated that, for the first time, the IOF-based dip-in indicator is able to detect a slight difference in the fuel mixture composition (i.e., 0.4% of oil content). Moreover, the color/no-color differentiation platform is simple, powerful, and easy-to-read. This platform makes the dip-in indicator a promising tool for authentication and determination of fuel composition at the point-of-purchase or point-of-use.

Challenges and Future Trends in DNA Microarray Analysis

Abstract Since its introduction, the DNA microarray platform has experienced a tremendous growth and currently it is a powerful tool used in various biological applications. However, it has not been embraced in the molecular diagnostic market as much as it was anticipated in the early days. The challenge comes from the skepticism on the reproducibility of the microarray data and on the reliability of the biological interpretations inferred. In addition, the microarray technology is suffering from strong competition by other diagnostic techniques. On the other hand, integration of various steps of the microarray assay into a harmonized and miniaturized handheld device suitable for point-of-care (POC) has been a goal for the microarray community. In this respect, significant progress has been achieved in coupling the DNA microarrays with efficient liquid manipulation microsystems as well as in developing novel technologies of supporting subsystems that well suits future POC microarray devices.

NanoHDA: A nanoparticle-assisted isothermal amplification technique for genotyping assays

Isothermal methods, such as helicase-dependent amplification (HDA), have an advantage over polymerase chain reaction for DNA amplification owing to their ease of operation. Here, we developed a new HDA method that is nanoparticle-assisted, termed nanoHDA. This method uses gold nanoparticles (AuNPs) to improve the sensitivity and specificity of the isothermal method. In HDA, the denaturation of DNA templates is mediated by helicases, but this method is limited by the low denaturation efficiency of helicases. In this report, AuNPs with preferential affinity for single-stranded DNA (ssDNA) were utilized to improve the denaturation efficiency of helicases. The same affinity property of nanoparticles can also enhance specificity by suppressing primer-dimer formation. This nanoHDA method was employed to genotype the KRAS gene in genomic DNA samples from colorectal cancer patients, as achieved by the hybridization of nanoHDA amplicons using the NanoBioArray chip.

Enhanced destabilization of mismatched DNA using gold nanoparticles offers specificity without compromising sensitivity for nucleic acid analyses

Here, we report a method that uses gold nanoparticles (AuNPs) to enhance the specificity of DNA hybridization without reducing its detection sensitivity. The conventional stringent wash method utilizes high-temperature/low-salt conditions to enhance the specificity of DNA hybridization-based assays. This method creates a destabilizing environment for base pairing that affects specific and nonspecific duplexes. Therefore, specificity is achieved at the expense of signal intensity or sensitivity. However, in the proposed wash method, AuNPs predominantly destabilize nonspecific duplexes, offering specificity without compromising sensitivity. This AuNP wash technique has proven to be effective in detecting single nucleotide polymorphisms (SNPs) in genomic samples even at room temperature in a CD-like NanoBioArray (CD-NBA) chip. This method is also robust with sequence variation and is compatible with multiplex DNA analyses on microarrays. Thus, the AuNP wash method could potentially be useful for improving the accuracy of DNA hybridization results.

High-Throughput DNA Array for SNP Detection of KRAS Gene Using a Centrifugal Microfluidic Device

Here, we describe detection of single nucleotide polymorphism (SNP) in genomic DNA samples using a NanoBioArray (NBA) chip. Fast DNA hybridization is achieved in the chip when target DNAs are introduced to the surface-arrayed probes using centrifugal force. Gold nanoparticles (AuNPs) are used to assist SNP detection at room temperature. The parallel setting of sample introduction in the spiral channels of the NBA chip enables multiple analyses on many samples, resulting in a technique appropriate for high-throughput SNP detection. The experimental procedure, including chip fabrication, probe array printing, DNA amplification, hybridization, signal detection, and data analysis, is described in detail.