Influence of sandwich-type DNA construction strategy and plasmonic metal on signal generated by SERS DNA sensors.

The DNA biosensors are powerful tools in the gene mutation or pathogens detection. That is why there are a lot of DNA detection strategies and methods. Here we present the insight on a slightly overlooked DNA detection technique, surface-enhanced Raman scattering (SERS). The present work is a summary of the influence of the plasmonic metal of the SERS substrate and strategy of the sandwich-type biosensor construction, simply the placement of the Raman reporter and mismatches, on the SERS signal enhancement. We found that, although in general there is an increase in the intensity of the SERS signal when the distance between the Raman scatterer and the SERS-active surface decreases, for this type of DNA SERS sensor a greater intensity of the measured Raman signal is usually observed when the Raman reporter is farther away from the plasmonic substrate. This is probably caused by a significant change in the hybridisation efficiency for the different structures of the sensor analysed due to some steric hindrances.

Attachment of Single-Stranded DNA to Certain SERS-Active Gold and Silver Substrates: Selected Practical Tips.

Layers formed from single-stranded DNA on nanostructured plasmonic metals can be applied as "working elements" in surface-enhanced Raman scattering (SERS) sensors used to sensitively and accurately identify specific DNA fragments in various biological samples (for example, in samples of blood). Therefore, the proper formation of the desired DNA layers on SERS substrates is of great practical importance, and many research groups are working to improve the process in forming such structures. In this work, we propose two modifications of a standard method used for depositing DNA with an attached linking thiol moiety on certain SERS-active structures; the modifications yield DNA layers that generate a stronger SERS signal. We propose: (i) freezing the sample when forming DNA layers on the nanoparticles, and (ii) when forming DNA layers on SERS-active macroscopic silver substrates, using ω-substituted alkanethiols with very short alkane chains (such as cysteamine or mercaptopropionic acid) to backfill the empty spaces on the metal surface unoccupied by DNA. When 6-mercapto-1-hexanol is used to fill the unoccupied places on a silver surface (as in experiments on standard gold substrates), a quick detachment of chemisorbed DNA from the silver surface is observed. Whereas, using ω-substituted alkanethiols with a shorter alkane chain makes it possible to easily form mixed DNA/backfilling thiol monolayers. Probably, the significantly lower desorption rate of the thiolated DNA induced by alkanethiols with shorter chains is due to the lower stabilization energy in monolayers formed from such compounds.

Surface Enhanced Raman Spectroscopy for DNA Biosensors-How Far Are We?

A sensitive and accurate identification of specific DNA fragments (usually containing a mutation) can influence clinical decisions. Standard methods routinely used for this type of detection are PCR (Polymerase Chain Reaction, and its modifications), and, less commonly, NGS (Next Generation Sequencing). However, these methods are quite complicated, requiring time-consuming, multi-stage sample preparation, and specially trained staff. Usually, it takes weeks for patients to obtain their results. Therefore, different DNA sensors are being intensively developed by many groups. One technique often used to obtain an analytical signal from DNA sensors is Raman spectroscopy. Its modification, surface-enhanced Raman spectroscopy (SERS), is especially useful for practical analytical applications due to its extra low limit of detection. SERS takes advantage of the strong increase in the efficiency of Raman signal generation caused by a local electric field enhancement near plasmonic (typically gold and silver) nanostructures. In this condensed review, we describe the most important types of SERS-based nanosensors for genetic studies and comment on their potential for becoming diagnostic tools.

SERS Studies of Adsorption on Gold Surfaces of Mononucleotides with Attached Hexanethiol Moiety: Comparison with Selected Single-Stranded Thiolated DNA Fragments.

The attachment of DNA strands to gold surfaces is performed in many devices, such as various DNA sensors. One of the standard methods used to immobilize DNA on gold surfaces involves two steps: the attachment of a thiol linker group (usually in the form of alkanethiol moiety) to the DNA strand, and the chemical reaction between the thiol-terminated DNA and the gold surface. Since thiols react chemically with the surface of gold substrates, forming very stable Au-S bonds, it is often assumed that the chemisorption on the gold surface of nucleotides with an attached thiol linker group leads to the formation of an order layer with the linking moieties relatively densely packed on the gold surface. In this contribution we show that chemisorption of thiolated mononucleotides does not occur according to this model. For example, the thiolated mononucleotide containing adenine strongly interacts with the gold surface via the adenine moiety. Moreover, bonding of the mononucleotide containing adenine to the gold surface is relatively similar to the bonding of adenine, and the main difference is that the adenine interacts with the gold surface mainly through the pyrimidine ring, while for adenine mononucleotide interaction via the imidazole ring also significantly contributes to the total bonding. A similar effect was observed for the mononucleotide containing cytosine, and the main difference between the interaction with the gold surface of cytosine and cytosine mononucleotide is that mononucleotide containing cytosine interacts with the gold surface to a significantly larger extend via the carboxylic group of the base. We also show that the structure of the layer formed on the gold surface by the thiolated mononucleotides may be significantly different than the structure of the layer formed by thiolated single-stranded DNA containing even as few as two bases.