Preliminary Data on a SERS-Responsive Sensor Based on Metallic Nanostructures Functionalized by Aptamers Specific for Arsenic

Arsenic, in the form of arsenate (As) and arsenite (As), is a toxic carcinogen widely distributed in aqueous environments in many parts of the world. Efficient arsenic sensors in terms of sensitivity, selectivity, speed and portability are urgently needed. The present research was focused on the development of a gold nanostructured sensor, functionalized with organic molecules able to selectively bind arsenic, for SERS detection. As organic molecule, we here selected oligonucleotide aptamers specific for arsenic recognition relying on previous studies on a 100-mer arsenic-binding DNA aptamer (Ars3), selected for its highest affinity and specificity to arsenate and arsenite. The aptamer Ars3 was previously used as such in its whole sequence and no attempt to optimize it, in terms of size and sensor efficiency, or to unravel its binding mechanism with arsenic has been carried out. Furthermore, even if in the previously proposed Ars3-based sensors a low detection limit for As was achieved, the assembly of the probe and the detection methodologies were in most cases very complex and not suitable for the development of portable in situ devices. Thus, in order to optimize Ars3 and investigate its interaction with arsenic, we here designed shorter DNA sequences cutting the 3’ and 5’ ends of the parent aptamer, and carried out spectroscopic and electrophoretic analysis together with arsenic-binding assays by using a suitably functionalized affinity resin. In addition, a specific SERS-responsive system with the Ars3 parent aptamer was considered in view of its application with the modified DNA sequences here proposed. Collected findings highlighted that the parent aptamer did not bind arsenic with high affinity. This was also in agreement with very recent results, published concomitantly with our studies, which stated that Ars3 was not able to bind As and that all the positive binding data using Ars3 were due to the use of gold supports (SPR chips or gold nanoparticles) which tightly bound arsenic. Based on these results, no arsenic-binding DNA aptamers are currently known, thus underlining the need for actual arsenic binding aptamers to be implemented for SERS sensors. Photonics and Photoactive Materials Materials Research Forum LLC Materials Research Proceedings 16 (2020) 46-55 https://doi.org/10.21741/9781644900710-6 47 Introduction Arsenic poisoning is caused by elevated levels of arsenic in the body. The dominant basis of arsenic poisoning is from ground water that naturally contains high concentration of arsenic [1]. Chronic ingestion or long-term exposure to arsenic can cause a variety of cancer forms, skin lesions, and cardiovascular diseases [1]. Elevated concentrations of arsenic in ground water have been found in many parts of the world and the maximum tolerable value of arsenic in drinkable water, in agreement with the World Health Organization, is 10 μg/L (ppb) [2]. It is generally recognized that inorganic arsenic is more toxic than organic one, and among inorganic arsenic, arsenic in oxidation state +3 (i.e., arsenite, derived from arsenous acid) is more toxic than that in the oxidation state +5 (i.e., arsenate, derived from arsenic acid) [1]. Currently, the most used analytical methodologies for the detection of arsenic at very low concentrations are Atomic Fluorescence Spectroscopy (AFS), Atomic Absorption Spectroscopy (AAS) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) [1],[3],[4]. These techniques are very efficient, but not suitable for in situ analysis, expensive, time consuming, and require chemical and physical pre-treatment of the samples. Therefore, it is essential not only to develop sensitive and selective analytical techniques for the detection of arsenic even in traces in the environment, but also to construct efficient arsenicspecific sensors in terms of speed and portability, especially for developing countries with highrisk contaminated soils. In this context, Surface-Enhanced Raman Scattering (SERS) guarantees high sensitivity and allows engineering tools of extremely reduced size. SERS can accomplish the speciation analysis for species with different oxidation states, with no need for any complex preparation of the sample, and has thus emerged as an extremely promising solution for in situ detection of arsenic in the field, particularly when coupled with portable/handheld Raman spectrometers [1],[5]. The present research was focused on the realization of a gold nanostructured sensor for SERS detection, functionalized with organic molecules able to selectively bind arsenic. As organic molecules, we here selected oligonucleotide aptamers specific for arsenic recognition. Aptamers are DNA/RNA oligonucleotides (ON) able to recognize with high affinity and selectivity a specific target based on their peculiar three-dimensional structures [6]. Aptamers can be selected from huge libraries of molecules containing randomly generated sequences, and specifically bind different targets, from proteins, viruses or whole cells to small molecules or ions [6],[7]. About 10 years ago a Korean group identified a 100-mer DNA aptamer (named Ars3) having high affinity and specificity for arsenate (As) and arsenite (As), with dissociation constants in the low nM range [8]. This arsenic-binding aptamer was selected from a random DNA library of 100-mer oligonucleotides which contained two constant regions at their ends as well as a 40 nt random region in the middle: GGT AAT ACG ACT CAC TAT AGG GAG ATA CCA GCT TAT TCA ATT-N40-AGA TAG TAA GTG CAA TCT. The aptamers were selected using affinity chromatography-based SELEX (Systematic Evolution of Ligands by EXponential enrichment), obtained by immobilizing arsenic on an agarose resin. Quantitative analyses of the aptamer candidates by SPR revealed Ars3 as the aptamer with the highest affinity to arsenate and arsenite. The specific affinity interactions of the aptamer to arsenic were verified against other heavy metals as negative controls, confirming its high specificity [8]. From this first work, Ars3 has been explored also in subsequent researches on arsenic sensors. In all cases, the aptamer was used as such, that is containing the random part plus the two flanking constant regions (which were generally removed) required for the PCR (polymerase chain reaction) step of the SELEX, and no attempt to optimize it, in terms of size and sensorPhotonics and Photoactive Materials Materials Research Forum LLC Materials Research Proceedings 16 (2020) 46-55 https://doi.org/10.21741/9781644900710-6 48 efficiency, or to elucidate its binding mechanism with arsenic has been ever described [9-17]. Furthermore, even if the detection limit for As sensors previously reported exploiting Ars3 was generally low, the assembly of the probe and the detection methodologies were very complex and not suitable for the development of portable in situ devices. Thus, in order to optimize Ars3 and further analyse its interaction with arsenic, we designed shorter DNA sequences, cutting in various way the 3’ and 5’ ends of the parent aptamer, and carried out spectroscopic and electrophoretic analyses together with arsenic-binding assays by using a suitably functionalized affinity resin. Moreover, a SERS-responsive system to Ars3 was ad hoc implemented in view of its application with the modified DNA sequences here proposed. Results and Discussion The detection procedure and the arsenic-binding aptamer Ars3 optimization were both addressed, designing a set of shorter oligonucleotide sequences starting from the original 100-mer, cutting its constant parts in various ways but leaving unaltered the central core of the sequence, which should contain the residues necessary for the specific recognition. From the analysis of all the designed sequences using m-fold program we selected three sequences (Ars40, Ars58 e Ars64, Fig. 1) potentially able to form hairpin-loop structures with good stability as the parent aptamer. Figure 1: Folding prediction of Ars3 and the derived shortened sequences obtained using the mfold program (settings: 150 mM NaCl, 5 mM MgCl2; folding temperature, 25 °C). Then, once obtained the set of the selected sequences, we analysed them by circular dichroism (CD) spectroscopy in two different buffers: the selection buffer originally used for Ars3 aptamer (named SB, 50 mM Hepes, 150 mM NaCl, 0.5 mM MgCl2, 0.5 mM CaCl2, pH 7.5) and PBS (phosphate buffered saline, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). All the sequences showed in both buffers almost the same conformational behaviour, with quite similar bands in their CD spectra, evidencing the formation of B-DNA duplex tracts – probably due to hairpin-loop structures (Fig. 2a). CD-melting curves showed the highest melting temperature (Tm) for the 100-mer ON and the lowest Tm value for the 40-mer (Fig. 2b). All the 20 Ars64