key: cord-0725242-eyobbio1
authors: Gutiérrez-Gálvez, Laura; del Caño, Rafael; Menéndez-Luque, Iris; García-Nieto, Daniel; Rodríguez-Peña, Micaela; Luna, Mónica; Pineda, Teresa; Pariente, Félix; García-Mendiola, Tania; Lorenzo, Encarnación
title: Electrochemiluminescent nanostructured DNA biosensor for SARS-CoV-2 detection
date: 2022-01-01
journal: Talanta
DOI: 10.1016/j.talanta.2021.123203
sha: 0e090b87e1d93b68affc7ca1828abb05e13887a5
doc_id: 725242
cord_uid: eyobbio1

This work focuses on the development of an electrochemiluminescent nanostructured DNA biosensor for SARS-CoV-2 detection. Gold nanomaterials (AuNMs), specifically, a mixture of gold nanotriangles (AuNTs) and gold nanoparticles (AuNPs), are used to modified disposable electrodes that serve as an improved nanostructured electrochemiluminescent platform for DNA detection. Carbon nanodots (CDs), prepared by green chemistry, are used as coreactants agents in the [Ru(bpy)(3)](2+) anodic electrochemiluminescence (ECL) and the hybridization is detected by changes in the ECL signal of [Ru(bpy)(3)](2+)/CDs in combination with AuNMs nanostructures. The biosensor is shown to detect a DNA sequence corresponding to SARS-CoV-2 with a detection limit of 514 aM.

Coronavirus disease 2019, popularly known as COVID-19, is an infectious illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). On March 2020, when the confirmed cases of infection exceeded 118.000 in 114 countries and 4.291 deaths had been reported, COVID-19 was declared a pandemic by the World Health Organization (WHO) [1] . Since then, this pathology has caused approximately more than 5.35 million deaths and 275 million cases worldwide, so it is an unprecedented challenge that all countries must face. Initially, a very low number of infection cases were reported due to lack of resources and incapacity to distinguish between COVID-19 and common flu [2] . However, today a great number of diagnostic methodologies for COVID-19 based on the detection of SARS-CoV-2 antigens (antigen tests) or anti-SARS-CoV-2 antibodies (serological tests), or the amplification of different parts of the virus genome are available.

Within this last group, quantitative reverse transcription polymerase chain reaction (RT-qPCR) stands out, which is considered the "gold standard" for COVID-19 diagnosis [3] .

Nevertheless, it is difficult to apply RT-qPCR as a mass screening technique due to its high cost, laboriousness and the need for expensive equipment and highly qualified personnel [4] . On the other hand, antigen tests are an interesting alternative for mass testing because of their low cost, portability and high specificity, but they are usually less sensitive than amplification-based methods [3] . Finally, serological tests are very sensitive, but they are also not suitable for mass diagnosis due to their cost and the need for long periods of time for data reporting and for trained personnel [5] . Taking into account all the limitations that these methodologies present, the development and through high-energy electron transfer reactions, when they return to the ground state [10] .

Therefore, this technique could be considered as a combination of electrochemical and chemiluminescence strategies, so ECL harbors the wonderful advantages of both techniques [11] such as wide dynamic range [10] , a spatial and temporal control of light emission, a greater selectivity due to the possibility of controlling the generation of excited states by varying the potentials applied to the electrode and non-existent background noise as no excitation light sources are used [12] . ECL may occur through the annihilation pathway or the coreactant pathway. Unlike ion annihilation ECL, in which the excited state material is reached through the reaction between the radicals generated by the same chemical species (known as luminophore), coreactant ECL needs two different precursors (luminophore and coreactant) [12] . Although several coreactants have been described, the search for new coreactants that provide improved ECL is a great deal of interest.

Recently it has been described in the literature that the analytical properties of diagnostic tests (such as specificity, selectivity, or sensitivity) can be improved by introducing nanomaterials into the device design [2] . In this context, the use of carbon nanodots (CDs), a novel cerodimensional nanomaterial, in the ECL technique has attracted much attention because they have recently been revealed to be excellent coreactants [13] due to their high charge transfer efficiency [14, 15] . In addition, CDs have excellent optical properties and are biocompatible [16] . They are nanoestructures below 10 nm size and consists of a skeleton made on elements such as carbon, hydrogen, nitrogen and oxygen with a quasi-spherical shape and a set of surface functional groups (OH, COOH, NH2, CONH2) that improve its solubility in water and fluorescence [17] .

On the other hand, another type of nanomaterial that have attracted the scientific interest since many years ago are gold nanomaterials. They can have a wide variety of forms and shapes (spheres, rods or triangular among others) and are considered one of the most popular nanomaterial at sensor application due to their ease of synthesis and modification, large surface area, good biocompatibility and high stability. Hence, they are a perfect choice as transducers for biosensors. In this sense, we can find several works using spherical or other shape gold nanoparticle for DNA biosensors [7, [18] [19] [20] but as far as we know there have not been reported the use of mixtures of gold nanomaterials (AuNMs) of different shapes (gold nanoparticles (AuNPs) and gold nanotriangles (AuNTs)) as platforms for DNA biosensor development.

Hence, in this work we present for the first time the combination of two different nanomaterials, gold nanomaterials (AuNMs) and CDs for the development of an improved ECL DNA biosensor. The combination of both nanomaterials produces a synergic effect that enhance the ECL response, providing an efficient SARS-CoV-2 biosensing platform, based on the detection of its genetic code. SARS-CoV-2 can be detected by using target genes of the specific viral gene regions coding for the spike protein (S), nucleocapsid protein (N), the RNA-dependent RNA polymerase ( 

Thiol-probe 5'-SH-C6H10-CCATAACCTTTCCACATACCGCAGACGG -3' ProbeORF-SH Thiol-probe TAMRA 5'-SH-C6H10-CCATAACCTTTCCACATACCGCAGACGG - TAMRA-3' ProbeORFTAMRA- SH Complementary 5'-CCGTCTGCGGTATGTGGAAAGGTTATGG -3' SARS-CoV-2C Non-Complementary 5'-GACCGTCGAAGTAAAGGGTTCCATA -3' SARS-CoV-2NC

Green synthesis of CDs was carried out in a CEM Discover LabMate™ microwave synthesis reactor. The purification of the CDs was carried out using 0.45 µm nylon syringe filters and dialysis membranes (Spectra/Por® 6, MWCO, 1 KDa) provided by General Laboratory Supplies (SGL).

Metrohm screen-printed gold electrodes (AuSPE, DRP-220BT), which integrate a gold working electrode, a silver pseudoreference electrode and a gold auxiliary electrode.

Electrochemical measurements were carried out using a screen-printed electrode connector (Metrohm) as interface and Autolab (PGSTAT 30 potentiostat) from Metrohm.

Software package GPES 4.9 was used with the for the total control of the experiments and data acquisition.

J o u r n a l P r e -p r o o f WSxM software [21] was used for the acquisition and processing of images.

An incident light microscope Axioskop 2 MAT (ZEISS) Fluorescence microscope with a mercury short arc lamp HBO 50 W/AC L1 (OSRAM) was used for fluorescence microscopy experiments.

All material and solutions were sterilized in a Nüve OT012 autoclave before to be used.

CDs were synthesized as follows: 3 mL of tiger nut milk was treated in a quartz flask under magnetic stirring for 30 min at 200 ºC using a microwave reactor maintaining a 150 W constant power and 170 psi pressure. The yellow solution obtained was kept to room temperature, filtered with 0.45 µm nylon syringe filter and dialyzed for 90 minutes to purify the CDs. Finally, they were stored at 4 ºC and protected from light.

The synthesis of gold nanomaterials (AuNMs), mixture of gold nanotriangles (AuNTs) and gold nanoparticles (AuNPs), were carried out following a seed-mediated growth procedure [22] and based on the use of Na2S2O3 for HAuCl4 reduction. Summarily, gold J o u r n a l P r e -p r o o f nanomaterials (AuNMs) were prepared by adding 25 ml of 2 mM HAuCl4 to 30 mL of 0.5 mM Na2S2O3 under vigorous stirring. Around 10 min later, 12.5 mL of Na2S2O3 (0.5 mM) was added to the solution under vigorous stirring. Firstly, the solution turned clear and brown and finally deep red. During the first Na2S2O3 addition, the seeds are formed and in the second addition the growth of seeds and gold nanomaterials (nanotriangles and nanoparticles) formation occurs. Finally, the solution is maintained stirring for 45 min. A mixture of spherical and triangular gold nanoparticles is obtained.

Stock solution of the thiol-modified probe were prepared following the protocol for thiol- and stored at -20 ºC.

AuSPEs were activated by applying ten successive cyclic potential scans from -0. were incubated with the thiolated probe on the electrode surface. Then, the hybridization process described above was carried out. Next, the electrodes were washed with water to remove non-adsorbed material and the ECL signal was recorded as it is described above.

Finally, the SARS-CoV-2C concentration was calculated from the ECL signal average J o u r n a l P r e -p r o o f

We have previously reported the use of [Ru(bpy)3] 2+ /CDs system to detect DNA hybridization event, using electrochemiluminescent (ECL) transduction [23] . oxidation peak increases concomitantly as the cathodic peak disappears. The effect is more evident on increasing the CDs concentration (see Figure 1SI ), when a fixed [13] . The proposed mechanism is showed in Figure 2SI .

In the present work we have decided to go a step forward and combine the [Ru(bpy)3] 2+ /CDs system with gold nanomaterials (AuNMs) to improve the ECL response for the development of a selective and sensitive SARS-CoV-2 biosensor.

AuNMs serve as platform for the thiolated DNA probe immobilization as well as an efficient electrochemical platform.

Therefore, the first step in this work has focused on the synthesis and characterization of the nanomaterials, CDs and AuNMs.

CDs were synthesized by tiger nut milk carbonization and using hydrothermal treatments in microwave reactor synthesizer, as it is explained in detail in the experimental section.

Best results, considering the balance of speed and performance, were obtained with a reaction time of 30 min at a temperature of 200 ⁰ C, maintaining a constant pressure of Gold nanomaterials (AuNMs), a mixture of gold nanotriangles (AuNTs) and gold nanoparticles (AuNPs), were prepared through the seed-mediated growth method using Na2S2O3 and HAuCl4 as precursors, as is described in detail in Experimental section. As can be observed in Figure 1A , the presence of two bands at 1015 and 539 nm in the UVvisible-NIR, is associated with the formation of triangular nanoparticles (1015 nm) and spherical (539 nm) nanoparticles and confirms gold nanomaterial preparation.

The obtained AuNMs were characterized by different techniques as AFM, SEM and TEM to determine their size and morphology properties. The dimensions and morphology of the AuNMs were estimated by TEM. As can be observed in Figure 1B AuNMs do not show any morphology (see Figure 4SI ). Therefore, we can confirm the successful synthesis of the AuNMs, based on the results obtained above. 

Scheme 1 shows the development of the nanostructured electrochemiluminescent (ECL) DNA biosensor. We have prepared an efficient and simple ECL platform based on the use of AuNMs as modifiers of gold screen-printed electrodes (AuSPEs) and further immobilization of the capture probe on these nanostructures, as platform to develop a selective and sensitive strategy to detect characteristics SARS-CoV-2 sequences. As can be seen in Scheme 1, the first step is surface modification of the AuSPE with AuNMs.

Then, the hybridization of the probe-target is conducted to detect a specific DNA sequence of SARS-CoV-2, by immobilizing the thiolated capture probe (a specific thiolated sequence complementary to the target sequence) on the AuNMs. Finally, a mixture of [Ru(bpy)3] 2+ /CDs was added to the solution and used as ECL system to detect and quantify SARS-CoV-2 characteristic DNA sequences.

To obtain a reproducible final device, each step of the biosensor development has been characterized by SEM, AFM, Cyclic Voltammetry (CV) and ECL. Figure Figure   2D ) show a current increase of the gold characteristic peaks, proving AuSPE modification with AuNMs. The electroactive surface area before and after electrode modification were found to be 0.0084 and 0.0165 cm 2 , respectively. This result suggests an electroactive area increment due to AuNMs immobilization on to electrode surface.

Blue line of Figure 2E and F, shows the Cyclic voltammograms and ECL signals, respectively, of the [Ru(bpy)3] 2+ /CDs system at AuSPE before and after modification with AuNMs. As can be seen, the modification of the electrode with gold nanostructures, specially with the mixture of nanostructures of different shape, produces an increase in the electrochemical and ECL signal compared to the observed for the bare electrode (black line). This can be explained by the increase in the relative surface area and the synergistic effect that nanostructures with different shapes cause in improving electron transfer, as demonstrated by comparing with the behaviour observed when using AuNTs or AuNPS alone (see red and green lines of Figure 2E and F). Therefore, these results demonstrate that the modification of electrodes with a combination of different shape metallic nanostructures gives rise to improved ECL platforms. In this sense, it is reported that assembled gold or silver nanoparticles on the electrode surface can efficiently catalyze and enhance the luminol ECL [25, 26] . Besides, other authors have reported also the use of heteronanoestructures as coreaction accelerator of luminol-O2 system for catalyzing the coreactant effect to generate more reactive radicals, which remarkably improved the ECL luminous efficiency of luminol [27] . Under these premises and according with the results obtained in this work, we believe that electrodes modified with the mixture of gold nanostructures give rise to very stable surfaces with improved conductivity and ECL responses, which is probably due to their acting as coreaction modified electrodes. Scan rate: 10 mVs -1 .

As we described above, to achieve the probe immobilization on the AuNMs surface we use a capture probe that is a complementary sequence modified on 5' extreme with a thiol (see Table 1 ). Thiolated capture probe immobilization on the AuNMs modified electrodes (ProbeORF-SH/AuNMs/AuSPE) was assessed by fluorescence microscopy. In this case, in order to follow the AuNMs/AuSPE probe immobilization, a thiolated probe modified also with a fluorophore (TAMRA or tetramethylrhodamine) (ProbeORFTAMRA-SH) was employed. As can be observed, white light optical microscope images before A) and after C) probe immobilization are quite similar. However, fluorescence images are very different since only the ProbeORFTAMRA-SH/AuNMs/AuSPE gives a fluorescence image, confirming that the DNA probe is bound to AuNMs/AuSPE ( Figure 3D ). As can be observed bare AuSPE modified with AuNMs do not show any fluorescence contrast (see Figure 3C ). ECL was employed to follow the biosensor response, as well as to confirm that the thiolated capture probe was successfully immobilized on the AuNMs modified electrodes. Hence, we studied the ECL of Ru(bpy)3] 2+ /CDs at a AuNMs/AuSPE and a ProbeORF-SH/AuNMs/AuSPE, (see Figure 3E ). As can be observed, after the probe immobilization there is an increase in the ECL signal compared to the small signal observed at AuNMs/AuSPE, which we believe is due to the coreactant effect of amine and hydroxyl groups present in the pirimidinic and purine bases. Therefore, these results are compatible with the capture probe immobilization on the AuNMs/AuSPE. 

Detection of SARS-CoV-2 was carried out by using [Ru(bpy)3] 2+ /CDs ECL system to detect the hybridization event between the probe and a specific sequence from open reading frame (ORF1ab) of the virus. After DNA probe immobilization on the AuNMs modified electrode, the hybridization with the target analyte was carried out on the resulting biosensor surface and detected by changes in the ECL signal of [Ru(bpy)3] 2+ /CDs system, enhanced by the presence of the AuNMs materials on the electrode surface. As can be observed in Figure 4A , biosensor response before hybridization (black bar) is higher than that observed after hybridization with the fully previously reported [28] .

The analytical parameters of the DNA biosensor were calculated under the optimal experimental conditions selected above. As can be observed in Figure 4B or Figure The selectivity of the biosensor to detect specific sequences SARS-CoV-2 was also evaluated. In particular, we have studied the variation of the ECL signal in samples containing SARS-CoV-2 sequence (50.0 pM) in the absence and in the presence of other virus sequences as Influenza A (H7N9) and SARS-CoV-1, at the same concentration (50.0 pM). In both cases, the ECL signal obtained is quite similar to those obtained for SARS-CoV-2 sequence (see Figure 5SI ). From these results, it can be concluded that the method can detect a target sequence of SARS-CoV-2 in presence of potential interfering sequences from other virus.

The reproducibility of the method was calculated using the response of three devices, prepared using the same protocol, fixing 50.0 pM as the analyte concentration sequence.

The relative standard deviation (RSD) was found to be to be 0.82% and a repeatability of 99.36% obtained by measuring 5 times the biosensor response. Furthermore, the biosensor can detect the target SARS-CoV-2 at least over a period of one month. 

The possibility of detecting SARS-CoV-2 variants is currently a great deal of interest.

Hence, we have studied the possibility of detecting gene mutation using the developed biosensor. In particular, Single Nucleotide Polymorphism (SNPs) detection have been studied. Figure 4A shows the ECL biosensor responses recorded after hybridization with a 1.0 nM solution of a single-mismatched (SARS-CoV-2SM) sequence (see Table 1 ). As can be seen, the biosensor response to the SNP containing sequence (SARS-CoV-2SM) produces a decrease ECL signal of 50 ± 7 a.u, which is much lower than the observed for the complementary sequence (SARS-CoV-2C) that produces a decrease of around 70 ± 5 a.u, confirming the high selectivity of the proposed biosensor.

Finally, we evaluated the applicability of the biosensor by determining SARS-CoV-2 sequences in human serum samples. J o u r n a l P r e -p r o o f Table 2 shows ECL or electrochemical DNA biosensors described in the literature to detect SARS-CoV-2 sequences. As can be observed, the analytical parameters of the developed biosensor compare well with those previously reported in the literature.

Moreover, the detection limit is comparable or even better and the developed biosensor is a simple, easy platform for SARS-CoV-2 detection. On the other hand, compared with other simple methodologies as colorimetric assay [33] , the proposed biosensor present 

The use of gold nanomaterials (AuNMs) in combination with [Ru(bpy)3] 2+ /CDs system has been proved as a new strategy to develop new electrochemiluminescent nanostructured biosensor for the selective and sensitive assay of SARS-CoV-2 virus DNA sequences. AuNMs allow the immobilization of the thiolated DNA capture probe, which recognized DNA sequences related to the virus. The hybridization detection is achieved by using [Ru(bpy)3] 2+ /CDs that, in combination with AuNMs, enhance the sensitivity of the platform. The system is able to detect not only SARS-CoV-2 sequences with a detection limit of 514 aM but also SNPs in the virus sequence.

This work has been financially supported by the Spanish Ministry of Economy and 

[1] WHO Director-General's opening remarks at the media briefing on COVID- 19 -11 March 2020 19 -11 March , (2020 . https://www.who.int/directorgeneral/speeches/detail/who-director-general-s-opening-remarks-at-the-mediabriefing-on-covid- 19---11-march-2020 19---11-march- (accessed June 11, 2021 . 

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