Quantitative Analysis of Intracellular Mirna Content Using Dual Gold and Iron Nanoreporters and Single Particle Icp-Tof-Ms

Quantitative Analysis of Intracellular Mirna Content Using Dual Gold and Iron Nanoreporters and Single Particle Icp-Tof-Ms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Quantitative Analysis of Intracellular Mirna Content Using Dual Gold and Iron Nanoreporters and Single Particle Icp-Tof-Ms Sara González-Morales, Elena Añón Alvarez, David Clases, Mario Corte-Rodriguez, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6279613/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Microchimica Acta → Version 1 posted 9 You are reading this latest preprint version Abstract MicroRNAs (miRNAs) are short single stranded RNA sequences that play an important role in the initiation and progression of cancer. Therefore, the present work tries to establish an analytical platform for the quantitative analysis of this miRNA in cancer cell models without enzymatic amplification reactions. The developed assay is based on a sandwich double-hybridization reaction using a capture oligonucleotide conjugated to magnetic iron oxide microparticles and a detection oligonucleotide conjugated to a 40 nm gold nanoparticle, both particles coated with streptavidin. The optimization of the double-hybridization assay is conducted using inductively coupled plasma in single particle mode with a time of flight analyzer (SP-ICP-ToF-MS) for double detection of Au and Fe within the same event. The developed strategy was directly applied to the quantification of miR-16-5p in cell lysates without amplification reactions. For this aim, the cancer cell line of melanoma (A375) was studied, and two sample preparation strategies have been evaluated. Sequence capturing in extracted RNA provided best results allowing the determination at about 200 pM of miR-16-5p (for 2x10 6 cells). This strategy represents one of the few alternatives to obtain absolute quantification of miRNA in biological samples to permit the direct comparison among cell lines without amplification or transformation reactions of the original sequence. miRNA cell lines Au/Fe simultaneous detection time of flight ICP-MS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION MicroRNAs (miRNAs) are small-sized transcripts that regulate expressions of genes at post-transcriptional level through specific targeting of mRNAs. With sizes about 21–25 nucleotides, miRNAs are produced in a multi-step process involving both nuclear and cytoplasmic proteins [ 1 ]. Some of these sequences are known to be involved in the carcinogenic process, since they regulate the expressions of several oncogenes and tumor suppressor genes as well as the activity of cancer-associated pathways [ 2 ][ 3 ]. In particular, miR-16-5p is an example of this class of structures with important roles in the development of diverse malignancies including neuroblastoma, osteosarcoma, hepatocellular carcinoma, breast cancer, gastrointestinal cancers, lung cancer and bladder cancer [ 4 ][ 5 ]. Specific studies in osteosarcoma cells have shown that the up-regulation of miR-16-5p suppressed proliferation, migratory potential and invasive features of this malignancy and increased the cytotoxic effects of cisplatin on these cells [ 6 ]. In breast cancer cell, down-regulation of miR-16-5p has been associated with a high migratory and proliferative potential of cells, an induction of cell cycle progression and a reduction of cell apoptosis [ 7 ]. Therefore, miRNAs have great potential as diagnostic or prognostic biomarkers but also as novel drugs or therapeutic targets. However, these promising clinical applications of miRNAs require methods to quantify expression levels accurately and reproducibly [ 8 ]. In general, quantification of miRNAs is possible by using hybridization, amplification, sequencing, and enzyme-based methods [ 9 , 10 ]. Real-time quantitative polymerase chain reaction (RT-qPCR) is considered a low to medium throughput method, suitable for the targeted quantification of miRNAs. However, variations in primer design, and inconsistent data analysis and normalization can negatively affect the reproducibility of RT-qPCR [ 11 ]. Nonetheless, due to its high sensitivity and specificity, RT-qPCR is the current gold standard method to verify data obtained by microarrays or next generation sequencing approaches. The use of metallic nanoparticles for miRNA detection represents also a growing trend. Spectroscopic detection of miRNA based on colorimetric methods [ 12 , 13 ] or light scattering [ 14 ] of gold nanoparticles have been reported. Recently, alternative strategies using such nanostructures as labels for elemental analysis using plasma mass spectrometry as detection method have been also implemented for miRNA or short RNA/DNA sequences in biological samples [ 15 – 17 ]. Previously, our group developed one of these platforms to address the presence of circulating miR-16-5p in blood serum by using SP-ICP-MS with gold nanoparticles as elemental labels [ 18 ]. That work presented the added value of mass spectrometric detection and was validated against RT-qPCR for the same set of samples with satisfactory quantitative results without amplification or transformation of the sought analyte. In this study, we attempted the analysis of miR-16-5p in cell cultures, which represents a more complex and demanding system. For this purpose, we have used a modified double hybrid sandwich assay and employed a new generation of ICP-MS equipped with a time of flight (ToF) mass analyzer. Such configuration permits the simultaneous monitoring of Au and Fe in the single event of the hybrid and avoids its disassembling, increasing the selectivity of the measurement. An instrument with a ToF analyzer provides new opportunities for single particle-based applications as they do not only have very fast spectra acquisition at 10 kHz and beyond but also record virtually any isotope across a mass range of 7–275 amu [ 19 – 21 ]. One important advantage of this platform is that on each individual experiment (calibration and sample), the analyte is recognized and captured using the same set of gold nanoparticles and magnetic Fe microparticles. As such, the stoichiometry of the number of probes per particle (nano or micro) does not need to be known. Similarly, gold nanoparticles transport efficiency to the ICP-MS does not need to be considered either, as every data point of the calibration curve and the sample will be affected by this efficiency in the same way [ 22 ]. However, a challenge is to increase the sensitivity of the assay aiming, in an ideal case, to “single molecule” detection by minimizing the number of probes per gold nanoparticle. Therefore, the optimization of the labelling of the Au-NPs with the probe is here carefully conducted using miR-16-5p as a model molecule. Under optimum conditions, the performance of the double hybrid sandwich assay using SP-ICP-To-MS is then applied for the determination of miR-16-5p in cell lysates of melanoma cell line using optimized sample preparation strategies and increased selectivity by using the simultaneous double detection of Fe and Au. This work aims to illustrate the possibilities of ICP-MS based bioassays for absolute miRNA quantification that can be extended to cells of different origin allowing comparative results using absolute concentration units. EXPERIMENTAL SECTION Instrumentation For the characterization of the assay via SP-ICP-ToF-MS, a Vitesse ICP-ToF-MS system by Nu Instruments (Wrexham, UK) was operated in single particle mode recording, binning (3 spectra) and saving mass spectra from 20 to 240 amu at 12.85 kHz (corresponding to a spectra saving interval of ~ 80 µs), while blanking the ranges 24.5–30.5 and 38–47 amu to avoid signal saturation at the detector. Data acquisition was performed by Nu Codaq Vitesse software (Nu Instruments) (version 1.5.8267.1), and SP ICP-ToF-MS raw data was directly processed by a modified version of SPCal (ver. 1.1.2), developed by Lockwood et al. [ 23 ][ 24 ] and adapted for ICP-ToF-MS data structure. Decision limits were determined using compound Poisson sampling of a lognormal approximation of the signal ion distribution. The plasma was operated at 1.35 kW and the segmented reaction cell was operated with helium (12 mL min − 1 ) and hydrogen (8 mL min − 1 ) as cell gases. Instrumental parameters such as nebulizer Ar flow and torch position were optimized daily to obtain the best sensitivity. For SP analysis, the ICP-ToF-MS system was equipped with a concentric nebulizer (Glass Expansion, Weilburg, Germany) and a cyclonic spray chamber. The nebulizer flow rate was tuned to provide the highest sensitivities while maintaining a CeO/Ce ratio below 10%. The aerosol's transport efficiency was determined by analyzing a diluted 80 nm Au NP standard (nanoComposix, California, US) and ionic standards of known concentration, using an automated approach via the SP data processing platform SPCal. Dilutions were performed in tubes made of polypropylene. Each sample, blank and calibration standard was recorded for 120 s. Operating conditions are shown in Table 1 . For spectrophotometric measurements of nucleic acid concentrations, a NanoDrop spectrophotometer (Thermo Fisher Scientific) was used. Other basic laboratory instrumentation was also used, including an analytical precision balance, an ultrasonic bath, a vortex mixer, and a block heater. Table 1 Operating conditions of the ICP-Tof-MS used in the study. REACTION CELL-OVERVIEW Parameters Au 50 nm Au 100 nm RF Set (V) 2.0 2.0 RF Power (W) 1350 1350 Peripump Speed (rpm) 50 50 Neb Flow (mL min − 1 ) 1150 1150 Entrance Aperture (V) -45.0 -45.0 Cell Entrance (V) -5.5 -3.0 Entrance offset (V) 6.0 5.0 Exit Offset (V) -12.0 -11.0 Cell Exit (V) -8.5 -10.0 Exit Aperture (V) -35.0 -30.0 Helium Flow (mL min − 1 ) 13.0 12.0 Hydrogen Flow (mL min − 1 ) 9.0 8.0 LOD (nm) 24.6 27.5 Materials and reagents All solutions were prepared in ultrapure water using a PURELAB flex 3 apparatus from Elga Veolia (High Wycombe, UK). For solutions needed to preserve miRNA samples, DEPC-treated water was purchased from Ambion and Life Technologies. Low protein binding microcentrifuge tubes from ThermoFisher Scientific were used to minimize the level of RNA-unspecific binding to plastic surfaces. Different gold nanoparticles have been used in this study: 1) the gold nanoparticles used for conjugation with the probe were 40 nm gold nanospheres coated with streptavidin OD10 from CD Bioparticles (New York, USA); 2) 80 nm Au NP standard from NanoComposix (San Diego, USA), characterized regarding size distribution, optical properties, surface potential and hydrodynamic radius by the manufacturer, to address transport efficiency in SP ICP-ToF-MS. Magnetic microparticles were SpeedBead Magnetic Streptavidin coated particles of 1 µm size from Nano Composix. Ionic gold 1000 ppm standard NIST3121 from Merck was used for calibration. All ionic solutions were diluted in 2% nitric acid prepared from 65% HNO 3 from Acros Organics (Geel, Belgium), previously purified by sub-boiling distillation. Argon gas for the operation of ICP-MS with 99.999% purity was supplied by Air Liquide (Paris, France). TRIS buffer saline (TBS) was purchased as a soluble tablet from Sigma-Aldrich. Phosphate buffer and aging buffer were prepared in-house using inorganic salts from Merck. Tween 20 was obtained from Sigma-Aldrich. TRIzol reagent from Invitrogen was used for the extraction of RNA from cells, in addition to chloroform, isopropanol and 75% ethanol in RNase-free water, all of them from Sigma-Aldrich. Elemental standards at 1000 µg L − 1 for ICP-MS (Single-Element ICP-Standard-Solution Roti®Star), diluted to working conditions using ultra-pure water (18.2 MΩ cm; Merck Millipore, Bedford, USA) were used. Oligonucleotides DNA oligonucleotides were custom-made by Invitrogen (Massachusetts, USA). They were shipped as a lyophilized powder and reconstituted as indicated by the manufacturer. Three DNA oligonucleotide sequences were used throughout this study: the target analyte miR-16-5p (1), the biotinylated half-complementary sequence to the 3′ end (capture probe) (2), and the also biotinylated half-complementary sequence to the 5′ end (detection probe) (3). All optimization steps were performed to avoid degradation problems associated with the manipulation of RNA using the equivalent DNA sequence for miR-16-5p (1*), since hybridization of RNA with DNA is also efficient. For this reason, detection and capture probes were kept as DNA even when using real miRNA samples. The complementary sequences were elongated with seven AAA triplets (total 21 A) for the capture probe and eight triplets (24 A) for the detection sequence. This elongation serves to separate the complementary region from the labeling group, avoiding any steric impairments. All sequences are specified in Table 2 . Table 2 DNA sequences used in the work of the target analyte, capture and detection probes. NAME SEQUENCE (1) miR-16-5p (target) 5’- UAG CAG CAC GUA AAU AUU GGC G -3’ (1)* miR-16-5p (surrogate DNA target) 5’- TAG CAG CAC GTA AAT ATT GGC G -3’ (2) Capture oligo (biotinylated) 5’- T TTA TAA CCG CAA (AAA) 7 -BIOT − 3’ (3) Detection oligo (biotinylated) 5’- BIOT-(AAA) 8 ATC GTC GTG CA -3’ Single Particle ICP-ToF-MS measurement (data acquisition and processing) SP-ICP-ToF-MS enables the rapid acquisition of full mass spectra, a critical requirement for fast non-target screenings of particulate elements [ 20 ]. However, its potential is partially limited by the generation of large data files that can reach several gigabytes per sample. To address this, we used SPCal software [ 24 ], which incorporates various tools for the processing of large SP-ICP-ToF-MS data. This involved multiple and iterative data processing steps, contributing to find SP signals, fit and smooth raw data, accumulate data points of individual SP signals, perform calibrations (e.g. mass, size, composition), calculate key parameters (e.g. limits of detection (LOD), transport efficiency, ionic response, etc.) and visualize data (histograms, charts, etc.). The characterization of the gold nanoparticles (40 nm) and validation of the assay was carried out by SP-ICP-ToF-MS. The ToF mass analyzer and ion optics enabled multi-element detection of events resulting from the ionization of individual particles by applying short integration times and high dilution factors. The signal intensity of the transient events caused by the single nanoparticles arriving at the ICP-ToF-MS was then transformed into the mass of gold by means of an external calibration curve of ionic gold and considering the transport efficiency of the ionic standards, which was calculated daily using the 80 nm Au NP and the ionic standards of known concentration. Once the mass of gold per nanoparticle was obtained, knowing the spherical geometry of the particles and their composition of pure gold, the volume and, therefore, the diameters were calculated. Preparation of the detection probe The detection probe was prepared by conjugating the detection (biotinylated) oligo with the 40-nm streptavidin-coated gold nanoparticles: 250 µ L of gold nanoparticles were precipitated by centrifugation at 10,000 rpm for 5 min, removing the supernatant and subsequently re-suspended in a TBS buffer containing 0.01% Tween 20. This suspension was mixed with 16.7 µ L of the detection biotinylated oligo and incubated for 30 min at room temperature. The freshly assembled detection probe was washed by centrifugation for 5 min at 10,000 rpm and re-suspended in 500 µ L of TBS. The number of washing steps was optimized to three. Preparation of the capture probe The capture probe was prepared by conjugating the capture biotinylated oligo with streptavidin-coated magnetic microparticles: 4 µ L of magnetic beads were washed three times using a magnet and a washing buffer with 2 M NaCl, 1 mM EDTA, and 10 mM Tris in ultrapure water at a pH of 7.5. 92 pmol of oligo were incubated with the microparticles for 20 min at room temperature. The excess oligo was then washed away with 2 washing steps using an external magnet to retain the conjugated magnetic microparticles. Cell cultures The A375 cell line was cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and 5 mg·L-1 Plasmocin. Cells were grown in T25 flasks within a humidified incubator maintained at 37°C and 5% CO 2 . The culture medium was refreshed every 2–3 days. When cells reached 80–90% confluency, they are subcultured. This involves removing the spent medium, washing the flask with PBS, and treating with 1 mL of 0.25% trypsin-EDTA for approximately 5 minutes to detach the cells from the flask surface. Following trypsinization, 5 mL of fresh DMEM was added to neutralize the trypsin and cells were pelleted by centrifugation at 400 g for 5 minutes. A 1:5 dilution of the cell suspension was then seeded into a new T25 flask. This process was repeated as needed to obtain the desired number of cells for the experiments. For every assay, the collected cells were counted using a Neubauer chamber. After trypsinization and pelleting by centrifugation, cells were appropriately diluted and 10 µL of this suspension was loaded into each chamber of the hemocytometer and observed under a microscope, counting viable cells within the four corner squares and the central square, consistently applying the same criteria for cells on the borders. Finally, cell concentration was calculated by averaging the number of cells per square, applying the corresponding dilution factor. Cell Lysis A cell lysis methodology was employed, based on freeze/thaw cycles to disrupt cell walls through osmosis and cyclic crystallization/melting. For this purpose, the treated cell pellet was resuspended in 1mL of Milli-Q water. Five freezing cycles in liquid nitrogen and thaw cycles in a 50°C water bath were performed on the cell suspension. Subsequently, the resulting solution was centrifuged for 10 minutes at 5,000 g, yielding a precipitate of cell debris and organelles, and a supernatant containing the analyte with other ionic compounds and proteins present in the cytosol. This extract was used as such to attempt the capturing of the analyte. Sample Pre-Treatment for RNA isolation Total cytosolic RNA was extracted from cultured cells using TRIzol reagent following standard procedures. Briefly, cells were harvested from culture flasks by trypsinization and pelleted by centrifugation at 400 g for 5 minutes. The cell pellet was then lysed in 1 mL of TRIzol reagent per 1-5x10⁶ cells, homogenized and incubated at room temperature for 5 minutes to dissociate nucleoprotein complexes. To separate phases, 0.2 mL of chloroform per 1 mL of TRIzol was added, followed by vigorous shaking and a 2–3-minute incubation at room temperature. After centrifugation at 12,000 ×g for 15 minutes at 4°C, the aqueous phase containing RNA was carefully transferred to a new tube, and RNA was precipitated by adding 0.5 mL of isopropanol per 1 mL of TRIzol. The sample was incubated at room temperature for 10 minutes, followed by centrifugation at 12,000 × g for 10 minutes at 4°C. The RNA pellet was washed with 75% ethanol, air-dried, and resuspended in 100 µ L RNase-free water. RNA concentration and purity were assessed by spectrophotometry at 260/280 and 260/230 nm, respectively. SP-ICP-ToF for characterization of the assay In a typical optimized assay, the capture and detection probes were freshly prepared the day before. 100 µL of the cell lysate were diluted to 500 µL in TBS. In the case of calibrations, the sample was a blood serum pool, which was fortified with the corresponding volume of surrogate DNA target sequence. Then, 26 µL of detection probe and 141 µL of capture probe were added and incubated at 70°C for 10 min to denature any hybridization or secondary structures of the probes or the analyte. This temperature was higher than the melting point of all the used oligos but lower than 80°C, which would have caused denaturation of the biotin. The mixture was then allowed to cool slowly to room temperature for 3 hours to guarantee specific hybridization, washed four times with TBS, using a magnet to collect the sandwich containing the magnetic beads and finally re-suspended in 300 µL of TBS. The resulting sandwich suspension was then adequately diluted and measured by SP-ICP-ToF-MS. RESULTS AND DISCUSSION Influence of the size of Au NPs and magnetic microparticles on the assay performance The scheme of the sandwich double hybridation assay is shown in Figure S1 . It is worth mentioning that the initial assay (optimized using a ICP-TQ-MS) included the use of thiolated Au-NPs with a nominal size of 25 nm [ 18 ]. However, the size detection limit obtained with the ICP-ToF-MS for gold nanoparticles, which turned out to be around 25 nm, did not allow their use. Therefore, an initial modification of the assay was carried out to use the new Au-NPs of larger diameter and containing streptavidin instead of thiolate groups. In this case, the recognition probe had to be equally modified to a sequence containing biotin instead of thiol groups at the end for conjugation with the NPs (Table 2 ). Therefore, the new system needed to be first characterized. In addition, when using SP-ICP-ToF-MS, the two elemental labels of the final product after the analyte was captured (Au and Fe) can be simultaneously monitored while this cannot be done when using SP-ICP-MS (where the disassembling of the sandwich assay before analysis was required). Such possibility allowed a more selective study of the blank contribution, since detected particles containing only gold or iron would not be forming the sandwich including the analyte. Thus, different sized Au-NPs were tested (40 and 60 nm), both coated with streptavidin, to be labelled with the biotinylated corresponding oligonucleotide to obtain the detection probe. The suitability of the instrument for the accurate measurements of both sized particles was tested using the conditions previously described for SP-ICP-ToF-MS (see Table 1 ). The size histograms (see Figure S2) showed a mean size of 40.7 ± 5.4 nm for the 40 nm Au NPs and 59 ± 13 nm for the 60 nm, which fitted well to the values provided by the manufacturers. Therefore, both particle suspensions were used as detection probes. Similarly, two different sized streptavidin-coated magnetic beads (1 and 2 µm, respectively) were tested for conjugation to the biotinylated oligonucleotide to produce the capture probe. Figure 1 (A, B and C) shows the obtained results for the different combinations of detection and capture probes under evaluation represented as the number of detected Au events (solid bar) and events containing both Au and Fe (dotted bar, Au + Fe events) versus increasing concentrations of the target miRNA. not hybridized with the analyte or the capture probe. Gold events that were coincident with iron could be ascribed to be corresponding to the sandwich formed by the detection probe, the miRNA and the capture probe. Only this combined Au + Fe events should be counted in the quantification of the analyte. In this regard, a small fraction of the gold events did also contain Fe (about 450 events in Fig. 1 A and up to 6000 events in Fig. 1 B) even in the absence of the analyte, ascribed to unspecific interactions between the two probes. The lowest blank levels were observed when applying 60 nm gold probes and 1 µM Fe-beads but no increasing response was observed (regarding Au + Fe events) upon varying the miRNA concentration. Finally, the combination of 40 nm gold probes and 1 µM Fe-beads was selected to perform further experiments. As can be seen, independent from the combination of detection/capture system used, there was a significant contribution of just gold containing events (solid part of the bars) even after thorough cleaning of the assay. These were only detection probes that were Fig. 2 shows the simultaneous presence of Fe (red trace) and Au (blue) in one of the events obtained applying the developed strategy to 1500 pM of the miRNA in the SP-ICP-ToF-MS revealing the capabilities of this type of instrumentation to discriminate between just Au or Au + Fe containing signals. This allows increasing selectivity and sensitivity of the assay with respect of measuring just Au. Optimization of the concentration of the oligo/NPs ratio for maximum sensitivity Aiming to increase the sensitivity of the assay, the minimum concentration of the detection oligonucleotide necessary to maintain the recognition capabilities in the detection probe for the analyte was evaluated. Two procedures were conducted: first, the concentration of Au NPs was kept constant while the concentration of oligo was reduced in successive experiments from the initial conditions and the response tested SP-ICP-ToF-MS monitoring events containing both Au and Fe. In a second experiment, the concentration of oligo was kept constant while the number of particles was reduced. In the first case, a linear response with the analyte concentration was obtained when halving the amount of detection probe (Figure S3). Lower concentrations of oligo showed similar blank values but a low linearity of the response against higher analyte concentrations. In the case of maintaining a constant concentration of the detection oligo and decreasing the number of particles, the results are shown in Fig. 3 . As can be seen, there are no significant changes in the linearity of the assays when decreasing the number of particles in the final assay from 1.6 x 10 11 (Fig. 3 A) to 3.2 x 10 10 (Fig. 3 D). Further dilutions showed a loss of linearity (not shown) and thus the final number of particles was adjusted to conditions used in Fig. 3 D and with a mass of oligo of about 400 pmol. It is worth mentioning that every data point of the calibration curves (in Figs. 3 and S3) is obtained in an independent assay. Nevertheless, the blank levels when comparing events containing gold and iron show remarkably similar values and always around 450–500 events. These results point out, on the one hand, the reproducibility of the assay and in another, the limitation to increase sensitivity affected by the unspecific blank signals that can not be lowered, even when different cleaning strategies were previously tested [ 18 ]. The limit of detection was calculated using these set of conditions and turned out to be about 160 pM of miRNA. Under optimum conditions, a study on the reproducibility of the assay at 1500 pM concentration of miRNA was done obtaining the results of Fig. 4 . The reproducibility among replicates is below 10%. This emphasizes the challenge of washing out the unreacted particles, but, on the other hand, shows the reproducibility also in the case of the blanks, which allows to subtract the blank signals from the sample signals when the assay is applied to real samples. These performance characteristics impulse the application of the strategy to the analysis of miR-16-5p in tumor cells without spiking or amplification. Application of the methodology to the analysis of miR-16-5p to cell cultures. As previously stated, miR-16-5p shows important roles in the development of diverse malignancies including neuroblastoma, osteosarcoma, hepatocellular carcinoma, etc. [ 25 ]. Therefore, its role as biomarker of these malignancies is well stablished [ 1 , 2 ]. Previous studies indicated that melanoma cell lines, specifically the model A375, is among the top 20 cell models expressing the sought miRNA and therefore, this cell line was selected to initiate the studies in real samples [ 26 ]. Two different sample preparation strategies were used, considering the potential presence of the analyte within the cell cytosol: 1) the cell lysis and direct analysis of miR-16-5p in the lysate and 2) isolation of total RNA using an established protocol and analysis of the sought sequence with the proposed strategy. For the first protocol, an ultrafiltration was performed after cell lysis so that the residual membranes and organelles were eliminated. This filtration step was mandatory since the direct assay on the cell lysate caused the aggregation of the nanoparticles (see Fig. S4). The obtained results are shown in Fig. 5 A for different numbers of lysed cells. Although the number of gold events was relatively high even with the lowest number of cells, the number of events containing both Au and Fe were below those observed for the blank of the assay (see Fig. 3 ). As previously observed during method development, this was likely due to the unspecific adsorption of the Au-probes to different molecules within the cytosol, reducing the capabilities of the capture probe to recover the miR-16-5p molecules. When increasing the number of cells, an increase of the number of events containing Au + Fe revealed the capabilities of the assay to reflect the increase in the miR-15-5p concentration. However, these levels were still below the calibration blank. Thus, an additional sample preparation step was conducted in order to selectively isolate RNA from the cells using the TRIZol protocol and then conduct the capturing from this extract. The purity of extracted RNA (obtained spectrophotometrically) was obtained for 2x10 6 , 6x10 6 and 12 x10 6 cells with a mean value (ratio 260/280 nm) of 1.93 (ratios of about ∼2.0 are considered as pure RNA). The results obtained for this assay are shown in Fig. 5 B. The number of Au events was comparable to those previously determined in the assay with standards (see Fig. 1 A) and the Au and Fe event number was always above the calibration blank allowing to quantify miR-16-5p even in the 2x10 6 cells extract which turned out to be about 200 pM (close to the calculated method DL). For the other samples, the results obtained corresponded to 427 pM (6x10 6 cells) and 636 pM (12 x10 6 cells). By triplicating the number of cells (from 2x10 6 to 6x10 6 ) the increase of the obtained concentration corresponded to about 70% of what was expected. Further increase in the cell number concentrations (from 6x10 6 to 12x10 6 ) yielded on an increase of about 75% with respect to what was expected. This could be also ascribed to the overall RNA extraction yields obtained for each experiment, which could be highly dependent on the number of cells. Therefore, the extraction of the miR-16-5p could be also affected by the inaccuracy of such procedure. In any case, the obtained results show adequate suitability for miR-16-5p quantification in the cell extracts to be used for comparative purpose among cell types. CONCLUSIONS The double hybrid sandwich assay using 40 nm Au-nanoparticles as detection probe and magnetic Fe microparticles as capture probe, both coated with streptavidin, provided good selectivity towards miR-16-5p. Eliminating the need for nucleic acid sequence amplification, our approach streamlined the analysis process while reducing potential sources of error that are common in conventional techniques. The optimization of the reagents permitted to maximize the stoichiometry aiming to improve the method sensitivity. In addition, the combination of the assay with the use of SP-ICP-ToF-MS as detector permits a double monitoring of Fe and Au, facilitating the confirmation of the hybrid formation and the discrimination of the contribution of the blanks due to unspecific adsorption of some of the probes. As such, restricting the detection to the condition that events must contain Fe and Au adds selectivity and sensitivity to the proposed assay. The final approach has been successfully tested to quantify the sought sequence in cell lysates of melanoma showing the possibility of absolute quantification after RNA extraction. Declarations FUNDING The authors gratefully acknowledge the financial support from the Spanish MICINN (Project Number PID2022-137222OB-I00) and from Principado de Asturias/Sekuens (Grant Number: IDE/2024/000742). Author Contribution All authors whose names appear on the submission contributed as follows:S.G. M. the acquisition, analysis, and interpretation of dataE.A.A. drafted the work or revised it critically for important intellectual contentD.C. made substantial contributions to the conception or design of the work and the acquisition, analysis, or interpretation of data as well as in the creation of new software used in the workM.C.R. made substantial contributions to the conception or design of the work and analysis and interpretation of data as well; approved the version to be published;M.M.B. made substantial contributions to the conception or design of the work and analysis and interpretation of data as well and drafted the work or revised it critically for important intellectual content Acknowledgement SGM acknowledges EMBO for the support for the stay (Scientific Exchange Grant 10518). References Diener C, Keller A, Meese E (2022) Emerging concepts of miRNA therapeutics: from cells to clinic. Trends Genet 38:613–626. https://doi.org/10.1016/j.tig.2022.02.006 Galvão-Lima LJ, Morais AHF, Valentim RAM, Barreto EJSS (2021) miRNAs as biomarkers for early cancer detection and their application in the development of new diagnostic tools. Biomed Eng Online 20:21. https://doi.org/10.1186/s12938-021-00857-9 Yang L, Yang S, Ren C et al (2022) Deciphering the roles of miR-16-5p in malignant solid tumors. Biomed Pharmacother 148:112703. https://doi.org/10.1016/j.biopha.2022.112703 Ghafouri-Fard S, Khoshbakht T, Hussen BM et al (2022) A review on the role of mir-16-5p in the carcinogenesis. Cancer Cell Int 22:342. https://doi.org/10.1186/s12935-022-02754-0 Hu H, Chen C, Chen F, Sun N (2022) LINC00152 knockdown suppresses tumorigenesis in non-small cell lung cancer via sponging miR-16-5p. J Thorac Dis 14:614–624. https://doi.org/10.21037/jtd-22-59 Gu Z, Li Z, Xu R et al (2020) miR-16-5p Suppresses Progression and Invasion of Osteosarcoma via Targeting at Smad3. https://doi.org/10.3389/fphar.2020.01324 . Front Pharmacol 11: Wang Z, Hu S, Li X et al (2021) MiR-16-5p suppresses breast cancer proliferation by targeting ANLN. BMC Cancer 21:1188. https://doi.org/10.1186/s12885-021-08914-1 Cissell KA, Deo SK (2009) Trends in microRNA detection. Anal Bioanal Chem 394:1109–1116. https://doi.org/10.1007/s00216-009-2744-6 Nelson PT, Baldwin DA, Scearce LM et al (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1:155–161. https://doi.org/10.1038/nmeth717 Hsu R-J, Yang H-J, Tsai H-J (2009) Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs. Nucleic Acids Res 37:e77–e77. https://doi.org/10.1093/nar/gkp274 Kalogianni DP, Kalligosfyri PM, Kyriakou IK, Christopoulos TK (2018) Advances in microRNA analysis. Anal Bioanal Chem 410:695–713. https://doi.org/10.1007/s00216-017-0632-z Shi H, Yang L, Zhou X et al (2017) A gold nanoparticle-based colorimetric strategy coupled to duplex-specific nuclease signal amplification for the determination of microRNA. Microchim Acta 184:525–531. https://doi.org/10.1007/s00604-016-2030-1 Li R-D, Yin B-C, Ye B-C (2016) Ultrasensitive, colorimetric detection of microRNAs based on isothermal exponential amplification reaction-assisted gold nanoparticle amplification. Biosens Bioelectron 86:1011–1016. https://doi.org/10.1016/j.bios.2016.07.042 Ren M, Wang S, Cai C et al (2016) A simple and sensitive resonance light scattering method based on aggregation of gold nanoparticles for selective detection of microRNA-21. RSC Adv 6:83078–83083. https://doi.org/10.1039/C6RA12366J Zhu Y-L, Lian Y-M, Wang J-K et al (2021) Highly Sensitive and Specific Mass Spectrometric Platform for miRNA Detection Based on the Multiple-Metal-Nanoparticle Tagging Strategy. Anal Chem 93:5839–5848. https://doi.org/10.1021/acs.analchem.1c00065 Xu X, Chen J, Li B et al (2019) Single particle ICP-MS-based absolute and relative quantification of E. coli O157 16S rRNA using sandwich hybridization capture. Analyst 144:1725–1730. https://doi.org/10.1039/C8AN02063A Zhang S, Han G, Xing Z et al (2014) Multiplex DNA Assay Based on Nanoparticle Probes by Single Particle Inductively Coupled Plasma Mass Spectrometry. Anal Chem 86:3541–3547. https://doi.org/10.1021/ac404245z González Morales S, López-Portugués C, Fernández-Sanjurjo M et al (2024) Amplification-Free Strategy for miRNA Quantification in Human Serum Using Single Particle ICP–MS and Gold Nanoparticles as Labels. Anal Chem. https://doi.org/10.1021/acs.analchem.4c01904 Bradley VC, Manard BT, Hendriks L et al (2024) Quantifying platinum binding on protein-functionalized magnetic microparticles using single particle-ICP-TOF-MS. Anal Methods 16:3192–3201. https://doi.org/10.1039/D4AY00268G Gonzalez de Vega R, Lockwood TE, Paton L et al (2023) Non-target analysis and characterisation of nanoparticles in spirits via single particle ICP-TOF-MS. J Anal Spectrom 38:2656–2663. https://doi.org/10.1039/D3JA00253E Lockwood TE, Gonzalez de Vega R, Du Z et al (2024) Strategies to enhance figures of merit in ICP-ToF-MS. J Anal Spectrom 39:227–234. https://doi.org/10.1039/D3JA00288H Degueldre C, Favarger PY, Wold S (2006) Gold colloid analysis by inductively coupled plasma-mass spectrometry in a single particle mode. Anal Chim Acta 555:263–268. https://doi.org/10.1016/j.aca.2005.09.021 Lockwood TE, Gonzalez de Vega R, Clases D (2021) An interactive Python-based data processing platform for single particle and single cell ICP-MS. J Anal Spectrom 36:2536–2544. https://doi.org/10.1039/D1JA00297J Lockwood TE, Schlatt L, Clases D (2025) SPCal – an open source, easy-to-use processing platform for ICP-TOFMS-based single event data. J Anal Spectrom 40:130–136. https://doi.org/10.1039/D4JA00241E Yang L, Yang S, Ren C et al (2022) Deciphering the roles of miR-16-5p in malignant solid tumors. Biomed Pharmacother 148:112703. https://doi.org/10.1016/j.biopha.2022.112703 Kavakiotis I, Alexiou A, Tastsoglou S et al (2022) DIANA-miTED: a microRNA tissue expression database. Nucleic Acids Res 50:D1055–D1061. https://doi.org/10.1093/nar/gkab733 Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTARYMATERIALfinal.docx GraphicalAbstract.pptx Cite Share Download PDF Status: Published Journal Publication published 02 Jun, 2025 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 08 Apr, 2025 Reviews received at journal 07 Apr, 2025 Reviews received at journal 05 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Editor assigned by journal 30 Mar, 2025 Submission checks completed at journal 30 Mar, 2025 First submitted to journal 21 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6279613","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440187007,"identity":"692c4e51-0bf4-4d86-aeee-0c50ac636bfb","order_by":0,"name":"Sara González-Morales","email":"","orcid":"","institution":"University of Oviedo","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"González-Morales","suffix":""},{"id":440187008,"identity":"4ef4269f-fc17-4af5-b039-b19476ff56bc","order_by":1,"name":"Elena Añón Alvarez","email":"","orcid":"","institution":"Instituto de Investigación Sanitaria del Principado de Asturias","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"Añón","lastName":"Alvarez","suffix":""},{"id":440187009,"identity":"02b3075d-fb42-4763-a97a-8ee773b08ac8","order_by":2,"name":"David Clases","email":"","orcid":"","institution":"University of Graz","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Clases","suffix":""},{"id":440187010,"identity":"0a5234e7-aa08-4674-b33c-7be8623667cd","order_by":3,"name":"Mario Corte-Rodriguez","email":"","orcid":"","institution":"University of Oviedo","correspondingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Corte-Rodriguez","suffix":""},{"id":440187011,"identity":"f4c29252-d040-45af-b726-9840ea37a984","order_by":4,"name":"Maria Montes-Bayon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFADZgbGBwwMEiAGQcDYANXCbECiFgYGNgminKPb3nv8wYc/2+TN25mfVfPusJAzOM7A+PAHHi1mZ84lNs5su2045zCb2W3eMxLGBocZmI158Gm5kWPYzNtwm3EGMwNQS5tE4sxmBjZpfA4Da/nz57b9DGb2b8VQLew/8ToMpIWB7XbiDGYeM2aQln5mBjYGvA47c8ZwZm/b7WSglmLJuW0SxvzMjM3SeLUc7zH48OPPbdsZ/Mc3fnjbVifHxn/44Ed8DsMG4BE1CkbBKBgFo4BcAAArqUfo/eaMNgAAAABJRU5ErkJggg==","orcid":"","institution":"University of Oviedo","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"","lastName":"Montes-Bayon","suffix":""}],"badges":[],"createdAt":"2025-03-21 17:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6279613/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6279613/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00604-025-07236-4","type":"published","date":"2025-06-02T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81054951,"identity":"ed70d6bb-eab6-43bd-b821-8c7dff2cd064","added_by":"auto","created_at":"2025-04-21 17:16:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":333178,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of events for the different combinations of detection and capture probes under evaluation represented as the number of detected Au events (solid bar) and events containing both Au and Fe (dotted bar, Au+Fe events) versus increasing concentrations of the miRNA under study. The combinations correspond to: A) 40 nm AuNPs + 1uM beads, B) 40nm AuNPs + 2uM beads and C) 60 nm Au NPs + 1uM beads.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/428d42428b57633df1246941.png"},{"id":81054952,"identity":"8110c9fe-4630-426f-91f4-94fba6e1b409","added_by":"auto","created_at":"2025-04-21 17:16:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56081,"visible":true,"origin":"","legend":"\u003cp\u003eType of events obtained for the hybrid combination containing the miRNA under study, the Au-probe and the Fe-probe using ICP-TOF-MS.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/f474fca09b8f682249394076.png"},{"id":81055571,"identity":"8821d39f-88d4-4dd0-b698-fd205cfdc70f","added_by":"auto","created_at":"2025-04-21 17:24:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":238653,"visible":true,"origin":"","legend":"\u003cp\u003eRegression lines obtained for the Au-Fe containing events obtained upon increasing the miRNA concentration (pM) for A) 1.6 x 10\u003csup\u003e11\u003c/sup\u003e\u0026nbsp;B) 1 x 10\u003csup\u003e11\u003c/sup\u003e\u0026nbsp;C) 7 x 10\u003csup\u003e10\u003c/sup\u003e\u0026nbsp;and D) 3.2 x 10\u003csup\u003e10\u0026nbsp; \u003c/sup\u003eparticles per mL.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/52e417fdd417e4b0dc4b08aa.png"},{"id":81054956,"identity":"77ad62be-891d-47c6-ad07-07a3f038243b","added_by":"auto","created_at":"2025-04-21 17:16:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":276522,"visible":true,"origin":"","legend":"\u003cp\u003eReproducibility of four independent replicates of the assay. Blue bars (Au events) corresponding to the blanks and grey bars (Au + Fe events) corresponding to independent replicates of 1500 pM.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/47f2b0e7ba9974556f7dc67d.png"},{"id":81054958,"identity":"fd67190b-dcc4-4472-b940-dc4b3bbdbe9c","added_by":"auto","created_at":"2025-04-21 17:16:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":214765,"visible":true,"origin":"","legend":"\u003cp\u003eResults obtained from cell extracts taking different number of cells A) in the crude cell extract and B) in the pooled extracted RNA. Solid bars (Au events) correspond to the blank and dotted bars (Au + Fe events) corresponding to the increasing amounts of the miRNA\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/71fbd54f277b6703d72f56bb.png"},{"id":84243143,"identity":"17ff5d4b-a0b8-4dd9-ba3a-367a354cf290","added_by":"auto","created_at":"2025-06-09 16:12:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1900899,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/e555cb7e-b2ad-4e70-afdc-ea05a05b4b89.pdf"},{"id":81054971,"identity":"4740d05e-4a4d-456b-a90c-840836a2b64f","added_by":"auto","created_at":"2025-04-21 17:16:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3149892,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYMATERIALfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/a56089a3568336708dd67466.docx"},{"id":81054961,"identity":"891760c7-01e2-4e37-89d9-36fd41238704","added_by":"auto","created_at":"2025-04-21 17:16:18","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":121118,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.pptx","url":"https://assets-eu.researchsquare.com/files/rs-6279613/v1/810ff8ea943e804187a04f4e.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eQuantitative Analysis of Intracellular Mirna Content Using Dual Gold and Iron Nanoreporters and Single Particle Icp-Tof-Ms\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMicroRNAs (miRNAs) are small-sized transcripts that regulate expressions of genes at post-transcriptional level through specific targeting of mRNAs. With sizes about 21\u0026ndash;25 nucleotides, miRNAs are produced in a multi-step process involving both nuclear and cytoplasmic proteins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Some of these sequences are known to be involved in the carcinogenic process, since they regulate the expressions of several oncogenes and tumor suppressor genes as well as the activity of cancer-associated pathways [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In particular, miR-16-5p is an example of this class of structures with important roles in the development of diverse malignancies including neuroblastoma, osteosarcoma, hepatocellular carcinoma, breast cancer, gastrointestinal cancers, lung cancer and bladder cancer [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Specific studies in osteosarcoma cells have shown that the up-regulation of miR-16-5p suppressed proliferation, migratory potential and invasive features of this malignancy and increased the cytotoxic effects of cisplatin on these cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In breast cancer cell, down-regulation of miR-16-5p has been associated with a high migratory and proliferative potential of cells, an induction of cell cycle progression and a reduction of cell apoptosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, miRNAs have great potential as diagnostic or prognostic biomarkers but also as novel drugs or therapeutic targets. However, these promising clinical applications of miRNAs require methods to quantify expression levels accurately and reproducibly [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn general, quantification of miRNAs is possible by using hybridization, amplification, sequencing, and enzyme-based methods [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Real-time quantitative polymerase chain reaction (RT-qPCR) is considered a low to medium throughput method, suitable for the targeted quantification of miRNAs. However, variations in primer design, and inconsistent data analysis and normalization can negatively affect the reproducibility of RT-qPCR [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Nonetheless, due to its high sensitivity and specificity, RT-qPCR is the current gold standard method to verify data obtained by microarrays or next generation sequencing approaches. The use of metallic nanoparticles for miRNA detection represents also a growing trend. Spectroscopic detection of miRNA based on colorimetric methods [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] or light scattering [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] of gold nanoparticles have been reported. Recently, alternative strategies using such nanostructures as labels for elemental analysis using plasma mass spectrometry as detection method have been also implemented for miRNA or short RNA/DNA sequences in biological samples [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Previously, our group developed one of these platforms to address the presence of circulating miR-16-5p in blood serum by using SP-ICP-MS with gold nanoparticles as elemental labels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. That work presented the added value of mass spectrometric detection and was validated against RT-qPCR for the same set of samples with satisfactory quantitative results without amplification or transformation of the sought analyte.\u003c/p\u003e \u003cp\u003eIn this study, we attempted the analysis of miR-16-5p in cell cultures, which represents a more complex and demanding system. For this purpose, we have used a modified double hybrid sandwich assay and employed a new generation of ICP-MS equipped with a time of flight (ToF) mass analyzer. Such configuration permits the simultaneous monitoring of Au and Fe in the single event of the hybrid and avoids its disassembling, increasing the selectivity of the measurement. An instrument with a ToF analyzer provides new opportunities for single particle-based applications as they do not only have very fast spectra acquisition at 10 kHz and beyond but also record virtually any isotope across a mass range of 7\u0026ndash;275 amu [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne important advantage of this platform is that on each individual experiment (calibration and sample), the analyte is recognized and captured using the same set of gold nanoparticles and magnetic Fe microparticles. As such, the stoichiometry of the number of probes per particle (nano or micro) does not need to be known. Similarly, gold nanoparticles transport efficiency to the ICP-MS does not need to be considered either, as every data point of the calibration curve and the sample will be affected by this efficiency in the same way [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, a challenge is to increase the sensitivity of the assay aiming, in an ideal case, to \u0026ldquo;single molecule\u0026rdquo; detection by minimizing the number of probes per gold nanoparticle. Therefore, the optimization of the labelling of the Au-NPs with the probe is here carefully conducted using miR-16-5p as a model molecule. Under optimum conditions, the performance of the double hybrid sandwich assay using SP-ICP-To-MS is then applied for the determination of miR-16-5p in cell lysates of melanoma cell line using optimized sample preparation strategies and increased selectivity by using the simultaneous double detection of Fe and Au. This work aims to illustrate the possibilities of ICP-MS based bioassays for absolute miRNA quantification that can be extended to cells of different origin allowing comparative results using absolute concentration units.\u003c/p\u003e"},{"header":"EXPERIMENTAL SECTION","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInstrumentation\u003c/h2\u003e \u003cp\u003eFor the characterization of the assay via SP-ICP-ToF-MS, a Vitesse ICP-ToF-MS system by Nu Instruments (Wrexham, UK) was operated in single particle mode recording, binning (3 spectra) and saving mass spectra from 20 to 240 amu at 12.85 kHz (corresponding to a spectra saving interval of ~\u0026thinsp;80 \u0026micro;s), while blanking the ranges 24.5\u0026ndash;30.5 and 38\u0026ndash;47 amu to avoid signal saturation at the detector. Data acquisition was performed by Nu Codaq Vitesse software (Nu Instruments) (version 1.5.8267.1), and SP ICP-ToF-MS raw data was directly processed by a modified version of SPCal (ver. 1.1.2), developed by Lockwood \u003cem\u003eet al.\u003c/em\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and adapted for ICP-ToF-MS data structure. Decision limits were determined using compound Poisson sampling of a lognormal approximation of the signal ion distribution. The plasma was operated at 1.35 kW and the segmented reaction cell was operated with helium (12 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and hydrogen (8 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) as cell gases. Instrumental parameters such as nebulizer Ar flow and torch position were optimized daily to obtain the best sensitivity.\u003c/p\u003e \u003cp\u003eFor SP analysis, the ICP-ToF-MS system was equipped with a concentric nebulizer (Glass Expansion, Weilburg, Germany) and a cyclonic spray chamber. The nebulizer flow rate was tuned to provide the highest sensitivities while maintaining a CeO/Ce ratio below 10%. The aerosol's transport efficiency was determined by analyzing a diluted 80 nm Au NP standard (nanoComposix, California, US) and ionic standards of known concentration, using an automated approach \u003cem\u003evia\u003c/em\u003e the SP data processing platform SPCal. Dilutions were performed in tubes made of polypropylene. Each sample, blank and calibration standard was recorded for 120 s. Operating conditions are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For spectrophotometric measurements of nucleic acid concentrations, a NanoDrop spectrophotometer (Thermo Fisher Scientific) was used. Other basic laboratory instrumentation was also used, including an analytical precision balance, an ultrasonic bath, a vortex mixer, and a block heater.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOperating conditions of the ICP-Tof-MS used in the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eREACTION CELL-OVERVIEW\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAu 50 nm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAu 100 nm\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRF Set (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRF Power (W)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1350\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePeripump Speed (rpm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNeb Flow (mL min\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEntrance Aperture (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-45.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-45.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCell Entrance (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEntrance offset (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExit Offset (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-12.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-11.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCell Exit (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-10.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eExit Aperture (V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-35.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-30.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHelium Flow (mL min\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHydrogen Flow (mL min\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLOD (nm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMaterials and reagents\u003c/h3\u003e\n\u003cp\u003eAll solutions were prepared in ultrapure water using a PURELAB flex 3 apparatus from Elga Veolia (High Wycombe, UK). For solutions needed to preserve miRNA samples, DEPC-treated water was purchased from Ambion and Life Technologies. Low protein binding microcentrifuge tubes from ThermoFisher Scientific were used to minimize the level of RNA-unspecific binding to plastic surfaces.\u003c/p\u003e \u003cp\u003eDifferent gold nanoparticles have been used in this study: 1) the gold nanoparticles used for conjugation with the probe were 40 nm gold nanospheres coated with streptavidin OD10 from CD Bioparticles (New York, USA); 2) 80 nm Au NP standard from NanoComposix (San Diego, USA), characterized regarding size distribution, optical properties, surface potential and hydrodynamic radius by the manufacturer, to address transport efficiency in SP ICP-ToF-MS. Magnetic microparticles were SpeedBead Magnetic Streptavidin coated particles of 1 \u0026micro;m size from Nano Composix.\u003c/p\u003e \u003cp\u003eIonic gold 1000 ppm standard NIST3121 from Merck was used for calibration. All ionic solutions were diluted in 2% nitric acid prepared from 65% HNO\u003csub\u003e3\u003c/sub\u003e from Acros Organics (Geel, Belgium), previously purified by sub-boiling distillation. Argon gas for the operation of ICP-MS with 99.999% purity was supplied by Air Liquide (Paris, France). TRIS buffer saline (TBS) was purchased as a soluble tablet from Sigma-Aldrich. Phosphate buffer and aging buffer were prepared in-house using inorganic salts from Merck. Tween 20 was obtained from Sigma-Aldrich. TRIzol reagent from Invitrogen was used for the extraction of RNA from cells, in addition to chloroform, isopropanol and 75% ethanol in RNase-free water, all of them from Sigma-Aldrich.\u003c/p\u003e \u003cp\u003eElemental standards at 1000 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for ICP-MS (Single-Element ICP-Standard-Solution Roti\u0026reg;Star), diluted to working conditions using ultra-pure water (18.2 MΩ cm; Merck Millipore, Bedford, USA) were used.\u003c/p\u003e\n\u003ch3\u003eOligonucleotides\u003c/h3\u003e\n\u003cp\u003eDNA oligonucleotides were custom-made by Invitrogen (Massachusetts, USA). They were shipped as a lyophilized powder and reconstituted as indicated by the manufacturer. Three DNA oligonucleotide sequences were used throughout this study: the target analyte miR-16-5p (1), the biotinylated half-complementary sequence to the 3\u0026prime; end (capture probe) (2), and the also biotinylated half-complementary sequence to the 5\u0026prime; end (detection probe) (3). All optimization steps were performed to avoid degradation problems associated with the manipulation of RNA using the equivalent DNA sequence for miR-16-5p (1*), since hybridization of RNA with DNA is also efficient. For this reason, detection and capture probes were kept as DNA even when using real miRNA samples.\u003c/p\u003e \u003cp\u003eThe complementary sequences were elongated with seven AAA triplets (total 21 A) for the capture probe and eight triplets (24 A) for the detection sequence. This elongation serves to separate the complementary region from the labeling group, avoiding any steric impairments. All sequences are specified in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDNA sequences used in the work of the target analyte, capture and detection probes.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNAME\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSEQUENCE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(1) miR-16-5p (target)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;- UAG CAG CAC GUA AAU AUU GGC G -3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(1)* miR-16-5p (surrogate DNA target)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;- TAG CAG CAC GTA AAT ATT GGC G -3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(2) Capture oligo (biotinylated)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;- T TTA TAA CCG CAA (AAA)\u003csub\u003e7\u003c/sub\u003e -BIOT \u0026minus;\u0026thinsp;3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(3) Detection oligo (biotinylated)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026rsquo;- BIOT-(AAA)\u003csub\u003e8\u003c/sub\u003e ATC GTC GTG CA -3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eSingle Particle ICP-ToF-MS measurement (data acquisition and processing)\u003c/h3\u003e\n\u003cp\u003eSP-ICP-ToF-MS enables the rapid acquisition of full mass spectra, a critical requirement for fast non-target screenings of particulate elements [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, its potential is partially limited by the generation of large data files that can reach several gigabytes per sample. To address this, we used SPCal software [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which incorporates various tools for the processing of large SP-ICP-ToF-MS data. This involved multiple and iterative data processing steps, contributing to find SP signals, fit and smooth raw data, accumulate data points of individual SP signals, perform calibrations (e.g. mass, size, composition), calculate key parameters (e.g. limits of detection (LOD), transport efficiency, ionic response, etc.) and visualize data (histograms, charts, etc.).\u003c/p\u003e \u003cp\u003eThe characterization of the gold nanoparticles (40 nm) and validation of the assay was carried out by SP-ICP-ToF-MS. The ToF mass analyzer and ion optics enabled multi-element detection of events resulting from the ionization of individual particles by applying short integration times and high dilution factors. The signal intensity of the transient events caused by the single nanoparticles arriving at the ICP-ToF-MS was then transformed into the mass of gold by means of an external calibration curve of ionic gold and considering the transport efficiency of the ionic standards, which was calculated daily using the 80 nm Au NP and the ionic standards of known concentration. Once the mass of gold per nanoparticle was obtained, knowing the spherical geometry of the particles and their composition of pure gold, the volume and, therefore, the diameters were calculated.\u003c/p\u003e\n\u003ch3\u003ePreparation of the detection probe\u003c/h3\u003e\n\u003cp\u003eThe detection probe was prepared by conjugating the detection (biotinylated) oligo with the 40-nm streptavidin-coated gold nanoparticles: 250 \u003cem\u003e\u0026micro;\u003c/em\u003eL of gold nanoparticles were precipitated by centrifugation at 10,000 rpm for 5 min, removing the supernatant and subsequently re-suspended in a TBS buffer containing 0.01% Tween 20. This suspension was mixed with 16.7 \u003cem\u003e\u0026micro;\u003c/em\u003eL of the detection biotinylated oligo and incubated for 30 min at room temperature. The freshly assembled detection probe was washed by centrifugation for 5 min at 10,000 rpm and re-suspended in 500 \u003cem\u003e\u0026micro;\u003c/em\u003eL of TBS. The number of washing steps was optimized to three.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of the capture probe\u003c/h2\u003e \u003cp\u003eThe capture probe was prepared by conjugating the capture biotinylated oligo with streptavidin-coated magnetic microparticles: 4 \u003cem\u003e\u0026micro;\u003c/em\u003eL of magnetic beads were washed three times using a magnet and a washing buffer with 2 M NaCl, 1 mM EDTA, and 10 mM Tris in ultrapure water at a pH of 7.5. 92 pmol of oligo were incubated with the microparticles for 20 min at room temperature. The excess oligo was then washed away with 2 washing steps using an external magnet to retain the conjugated magnetic microparticles.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell cultures\u003c/h3\u003e\n\u003cp\u003eThe A375 cell line was cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and 5 mg\u0026middot;L-1 Plasmocin. Cells were grown in T25 flasks within a humidified incubator maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture medium was refreshed every 2\u0026ndash;3 days. When cells reached 80\u0026ndash;90% confluency, they are subcultured. This involves removing the spent medium, washing the flask with PBS, and treating with 1 mL of 0.25% trypsin-EDTA for approximately 5 minutes to detach the cells from the flask surface. Following trypsinization, 5 mL of fresh DMEM was added to neutralize the trypsin and cells were pelleted by centrifugation at 400 g for 5 minutes. A 1:5 dilution of the cell suspension was then seeded into a new T25 flask. This process was repeated as needed to obtain the desired number of cells for the experiments.\u003c/p\u003e \u003cp\u003eFor every assay, the collected cells were counted using a Neubauer chamber. After trypsinization and pelleting by centrifugation, cells were appropriately diluted and 10 \u0026micro;L of this suspension was loaded into each chamber of the hemocytometer and observed under a microscope, counting viable cells within the four corner squares and the central square, consistently applying the same criteria for cells on the borders. Finally, cell concentration was calculated by averaging the number of cells per square, applying the corresponding dilution factor.\u003c/p\u003e\n\u003ch3\u003eCell Lysis\u003c/h3\u003e\n\u003cp\u003eA cell lysis methodology was employed, based on freeze/thaw cycles to disrupt cell walls through osmosis and cyclic crystallization/melting. For this purpose, the treated cell pellet was resuspended in 1mL of Milli-Q water. Five freezing cycles in liquid nitrogen and thaw cycles in a 50\u0026deg;C water bath were performed on the cell suspension. Subsequently, the resulting solution was centrifuged for 10 minutes at 5,000 g, yielding a precipitate of cell debris and organelles, and a supernatant containing the analyte with other ionic compounds and proteins present in the cytosol. This extract was used as such to attempt the capturing of the analyte.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSample Pre-Treatment for RNA isolation\u003c/h2\u003e \u003cp\u003eTotal cytosolic RNA was extracted from cultured cells using TRIzol reagent following standard procedures. Briefly, cells were harvested from culture flasks by trypsinization and pelleted by centrifugation at 400 g for 5 minutes. The cell pellet was then lysed in 1 mL of TRIzol reagent per 1-5x10⁶ cells, homogenized and incubated at room temperature for 5 minutes to dissociate nucleoprotein complexes. To separate phases, 0.2 mL of chloroform per 1 mL of TRIzol was added, followed by vigorous shaking and a 2\u0026ndash;3-minute incubation at room temperature. After centrifugation at 12,000 \u0026times;g for 15 minutes at 4\u0026deg;C, the aqueous phase containing RNA was carefully transferred to a new tube, and RNA was precipitated by adding 0.5 mL of isopropanol per 1 mL of TRIzol. The sample was incubated at room temperature for 10 minutes, followed by centrifugation at 12,000 \u0026times; g for 10 minutes at 4\u0026deg;C. The RNA pellet was washed with 75% ethanol, air-dried, and resuspended in 100 \u003cem\u003e\u0026micro;\u003c/em\u003eL RNase-free water. RNA concentration and purity were assessed by spectrophotometry at 260/280 and 260/230 nm, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSP-ICP-ToF for characterization of the assay\u003c/h2\u003e \u003cp\u003eIn a typical optimized assay, the capture and detection probes were freshly prepared the day before. 100 \u0026micro;L of the cell lysate were diluted to 500 \u0026micro;L in TBS. In the case of calibrations, the sample was a blood serum pool, which was fortified with the corresponding volume of surrogate DNA target sequence. Then, 26 \u0026micro;L of detection probe and 141 \u0026micro;L of capture probe were added and incubated at 70\u0026deg;C for 10 min to denature any hybridization or secondary structures of the probes or the analyte. This temperature was higher than the melting point of all the used oligos but lower than 80\u0026deg;C, which would have caused denaturation of the biotin. The mixture was then allowed to cool slowly to room temperature for 3 hours to guarantee specific hybridization, washed four times with TBS, using a magnet to collect the sandwich containing the magnetic beads and finally re-suspended in 300 \u0026micro;L of TBS. The resulting sandwich suspension was then adequately diluted and measured by SP-ICP-ToF-MS.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of the size of Au NPs and magnetic microparticles on the assay performance\u003c/h2\u003e \u003cp\u003eThe scheme of the sandwich double hybridation assay is shown in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. It is worth mentioning that the initial assay (optimized using a ICP-TQ-MS) included the use of thiolated Au-NPs with a nominal size of 25 nm [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the size detection limit obtained with the ICP-ToF-MS for gold nanoparticles, which turned out to be around 25 nm, did not allow their use. Therefore, an initial modification of the assay was carried out to use the new Au-NPs of larger diameter and containing streptavidin instead of thiolate groups. In this case, the recognition probe had to be equally modified to a sequence containing biotin instead of thiol groups at the end for conjugation with the NPs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Therefore, the new system needed to be first characterized.\u003c/p\u003e \u003cp\u003eIn addition, when using SP-ICP-ToF-MS, the two elemental labels of the final product after the analyte was captured (Au and Fe) can be simultaneously monitored while this cannot be done when using SP-ICP-MS (where the disassembling of the sandwich assay before analysis was required). Such possibility allowed a more selective study of the blank contribution, since detected particles containing only gold or iron would not be forming the sandwich including the analyte. Thus, different sized Au-NPs were tested (40 and 60 nm), both coated with streptavidin, to be labelled with the biotinylated corresponding oligonucleotide to obtain the detection probe. The suitability of the instrument for the accurate measurements of both sized particles was tested using the conditions previously described for SP-ICP-ToF-MS (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The size histograms (see Figure S2) showed a mean size of 40.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 nm for the 40 nm Au NPs and 59\u0026thinsp;\u0026plusmn;\u0026thinsp;13 nm for the 60 nm, which fitted well to the values provided by the manufacturers. Therefore, both particle suspensions were used as detection probes.\u003c/p\u003e \u003cp\u003eSimilarly, two different sized streptavidin-coated magnetic beads (1 and 2 \u0026micro;m, respectively) were tested for conjugation to the biotinylated oligonucleotide to produce the capture probe. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (A, B and C) shows the obtained results for the different combinations of detection and capture probes under evaluation represented as the number of detected Au events (solid bar) and events containing both Au and Fe (dotted bar, Au\u0026thinsp;+\u0026thinsp;Fe events) versus increasing concentrations of the target miRNA. not hybridized with the analyte or the capture probe. Gold events that were coincident with iron could be ascribed to be corresponding to the sandwich formed by the detection probe, the miRNA and the capture probe.\u003c/p\u003e \u003cp\u003eOnly this combined Au\u0026thinsp;+\u0026thinsp;Fe events should be counted in the quantification of the analyte. In this regard, a small fraction of the gold events did also contain Fe (about 450 events in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and up to 6000 events in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) even in the absence of the analyte, ascribed to unspecific interactions between the two probes. The lowest blank levels were observed when applying 60 nm gold probes and 1 \u0026micro;M Fe-beads but no increasing response was observed (regarding Au\u0026thinsp;+\u0026thinsp;Fe events) upon varying the miRNA concentration. Finally, the combination of 40 nm gold probes and 1 \u0026micro;M Fe-beads was selected to perform further experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs can be seen, independent from the combination of detection/capture system used, there was a significant contribution of just gold containing events (solid part of the bars) even after thorough cleaning of the assay. These were only detection probes that were Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the simultaneous presence of Fe (red trace) and Au (blue) in one of the events obtained applying the developed strategy to 1500 pM of the miRNA in the SP-ICP-ToF-MS revealing the capabilities of this type of instrumentation to discriminate between just Au or Au\u0026thinsp;+\u0026thinsp;Fe containing signals. This allows increasing selectivity and sensitivity of the assay with respect of measuring just Au.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOptimization of the concentration of the oligo/NPs ratio for maximum sensitivity\u003c/h2\u003e \u003cp\u003eAiming to increase the sensitivity of the assay, the minimum concentration of the detection oligonucleotide necessary to maintain the recognition capabilities in the detection probe for the analyte was evaluated. Two procedures were conducted: first, the concentration of Au NPs was kept constant while the concentration of oligo was reduced in successive experiments from the initial conditions and the response tested SP-ICP-ToF-MS monitoring events containing both Au and Fe. In a second experiment, the concentration of oligo was kept constant while the number of particles was reduced. In the first case, a linear response with the analyte concentration was obtained when halving the amount of detection probe (Figure S3). Lower concentrations of oligo showed similar blank values but a low linearity of the response against higher analyte concentrations.\u003c/p\u003e \u003cp\u003eIn the case of maintaining a constant concentration of the detection oligo and decreasing the number of particles, the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. As can be seen, there are no significant changes in the linearity of the assays when decreasing the number of particles in the final assay from 1.6 x 10\u003csup\u003e11\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) to 3.2 x 10\u003csup\u003e10\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther dilutions showed a loss of linearity (not shown) and thus the final number of particles was adjusted to conditions used in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and with a mass of oligo of about 400 pmol. It is worth mentioning that every data point of the calibration curves (in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and S3) is obtained in an independent assay. Nevertheless, the blank levels when comparing events containing gold and iron show remarkably similar values and always around 450\u0026ndash;500 events. These results point out, on the one hand, the reproducibility of the assay and in another, the limitation to increase sensitivity affected by the unspecific blank signals that can not be lowered, even when different cleaning strategies were previously tested [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe limit of detection was calculated using these set of conditions and turned out to be about 160 pM of miRNA. Under optimum conditions, a study on the reproducibility of the assay at 1500 pM concentration of miRNA was done obtaining the results of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The reproducibility among replicates is below 10%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis emphasizes the challenge of washing out the unreacted particles, but, on the other hand, shows the reproducibility also in the case of the blanks, which allows to subtract the blank signals from the sample signals when the assay is applied to real samples. These performance characteristics impulse the application of the strategy to the analysis of miR-16-5p in tumor cells without spiking or amplification.\u003c/p\u003e \u003cp\u003e \u003cb\u003eApplication of the methodology to the analysis of miR-16-5p to cell cultures.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs previously stated, miR-16-5p shows important roles in the development of diverse malignancies including neuroblastoma, osteosarcoma, hepatocellular carcinoma, etc. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, its role as biomarker of these malignancies is well stablished [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous studies indicated that melanoma cell lines, specifically the model A375, is among the top 20 cell models expressing the sought miRNA and therefore, this cell line was selected to initiate the studies in real samples [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Two different sample preparation strategies were used, considering the potential presence of the analyte within the cell cytosol: 1) the cell lysis and direct analysis of miR-16-5p in the lysate and 2) isolation of total RNA using an established protocol and analysis of the sought sequence with the proposed strategy.\u003c/p\u003e \u003cp\u003eFor the first protocol, an ultrafiltration was performed after cell lysis so that the residual membranes and organelles were eliminated. This filtration step was mandatory since the direct assay on the cell lysate caused the aggregation of the nanoparticles (see Fig. S4). The obtained results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA for different numbers of lysed cells. Although the number of gold events was relatively high even with the lowest number of cells, the number of events containing both Au and Fe were below those observed for the blank of the assay (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As previously observed during method development, this was likely due to the unspecific adsorption of the Au-probes to different molecules within the cytosol, reducing the capabilities of the capture probe to recover the miR-16-5p molecules. When increasing the number of cells, an increase of the number of events containing Au\u0026thinsp;+\u0026thinsp;Fe revealed the capabilities of the assay to reflect the increase in the miR-15-5p concentration. However, these levels were still below the calibration blank.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThus, an additional sample preparation step was conducted in order to selectively isolate RNA from the cells using the TRIZol protocol and then conduct the capturing from this extract. The purity of extracted RNA (obtained spectrophotometrically) was obtained for 2x10\u003csup\u003e6\u003c/sup\u003e, 6x10\u003csup\u003e6\u003c/sup\u003e and 12 x10\u003csup\u003e6\u003c/sup\u003e cells with a mean value (ratio 260/280 nm) of 1.93 (ratios of about \u0026sim;2.0 are considered as pure RNA). The results obtained for this assay are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. The number of Au events was comparable to those previously determined in the assay with standards (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and the Au and Fe event number was always above the calibration blank allowing to quantify miR-16-5p even in the 2x10\u003csup\u003e6\u003c/sup\u003e cells extract which turned out to be about 200 pM (close to the calculated method DL). For the other samples, the results obtained corresponded to 427 pM (6x10\u003csup\u003e6\u003c/sup\u003e cells) and 636 pM (12 x10\u003csup\u003e6\u003c/sup\u003e cells). By triplicating the number of cells (from 2x10\u003csup\u003e6\u003c/sup\u003e to 6x10\u003csup\u003e6\u003c/sup\u003e) the increase of the obtained concentration corresponded to about 70% of what was expected. Further increase in the cell number concentrations (from 6x10\u003csup\u003e6\u003c/sup\u003e to 12x10\u003csup\u003e6\u003c/sup\u003e) yielded on an increase of about 75% with respect to what was expected. This could be also ascribed to the overall RNA extraction yields obtained for each experiment, which could be highly dependent on the number of cells. Therefore, the extraction of the miR-16-5p could be also affected by the inaccuracy of such procedure. In any case, the obtained results show adequate suitability for miR-16-5p quantification in the cell extracts to be used for comparative purpose among cell types.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe double hybrid sandwich assay using 40 nm Au-nanoparticles as detection probe and magnetic Fe microparticles as capture probe, both coated with streptavidin, provided good selectivity towards miR-16-5p. Eliminating the need for nucleic acid sequence amplification, our approach streamlined the analysis process while reducing potential sources of error that are common in conventional techniques. The optimization of the reagents permitted to maximize the stoichiometry aiming to improve the method sensitivity. In addition, the combination of the assay with the use of SP-ICP-ToF-MS as detector permits a double monitoring of Fe and Au, facilitating the confirmation of the hybrid formation and the discrimination of the contribution of the blanks due to unspecific adsorption of some of the probes. As such, restricting the detection to the condition that events must contain Fe and Au adds selectivity and sensitivity to the proposed assay. The final approach has been successfully tested to quantify the sought sequence in cell lysates of melanoma showing the possibility of absolute quantification after RNA extraction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThe authors gratefully acknowledge the financial support from the Spanish MICINN (Project Number PID2022-137222OB-I00) and from Principado de Asturias/Sekuens (Grant Number: IDE/2024/000742).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors whose names appear on the submission contributed as follows:S.G. M. the acquisition, analysis, and interpretation of dataE.A.A. drafted the work or revised it critically for important intellectual contentD.C. made substantial contributions to the conception or design of the work and the acquisition, analysis, or interpretation of data as well as in the creation of new software used in the workM.C.R. made substantial contributions to the conception or design of the work and analysis and interpretation of data as well; approved the version to be published;M.M.B. made substantial contributions to the conception or design of the work and analysis and interpretation of data as well and drafted the work or revised it critically for important intellectual content\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eSGM acknowledges EMBO for the support for the stay (Scientific Exchange Grant 10518).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDiener C, Keller A, Meese E (2022) Emerging concepts of miRNA therapeutics: from cells to clinic. Trends Genet 38:613\u0026ndash;626. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tig.2022.02.006\u003c/span\u003e\u003cspan address=\"10.1016/j.tig.2022.02.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalv\u0026atilde;o-Lima LJ, Morais AHF, Valentim RAM, Barreto EJSS (2021) miRNAs as biomarkers for early cancer detection and their application in the development of new diagnostic tools. Biomed Eng Online 20:21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12938-021-00857-9\u003c/span\u003e\u003cspan address=\"10.1186/s12938-021-00857-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Yang S, Ren C et al (2022) Deciphering the roles of miR-16-5p in malignant solid tumors. Biomed Pharmacother 148:112703. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2022.112703\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2022.112703\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhafouri-Fard S, Khoshbakht T, Hussen BM et al (2022) A review on the role of mir-16-5p in the carcinogenesis. Cancer Cell Int 22:342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12935-022-02754-0\u003c/span\u003e\u003cspan address=\"10.1186/s12935-022-02754-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu H, Chen C, Chen F, Sun N (2022) LINC00152 knockdown suppresses tumorigenesis in non-small cell lung cancer via sponging miR-16-5p. J Thorac Dis 14:614\u0026ndash;624. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21037/jtd-22-59\u003c/span\u003e\u003cspan address=\"10.21037/jtd-22-59\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu Z, Li Z, Xu R et al (2020) miR-16-5p Suppresses Progression and Invasion of Osteosarcoma via Targeting at Smad3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2020.01324\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2020.01324\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Front Pharmacol 11:\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Z, Hu S, Li X et al (2021) MiR-16-5p suppresses breast cancer proliferation by targeting ANLN. BMC Cancer 21:1188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12885-021-08914-1\u003c/span\u003e\u003cspan address=\"10.1186/s12885-021-08914-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCissell KA, Deo SK (2009) Trends in microRNA detection. Anal Bioanal Chem 394:1109\u0026ndash;1116. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-009-2744-6\u003c/span\u003e\u003cspan address=\"10.1007/s00216-009-2744-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNelson PT, Baldwin DA, Scearce LM et al (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods 1:155\u0026ndash;161. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth717\u003c/span\u003e\u003cspan address=\"10.1038/nmeth717\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsu R-J, Yang H-J, Tsai H-J (2009) Labeled microRNA pull-down assay system: an experimental approach for high-throughput identification of microRNA-target mRNAs. Nucleic Acids Res 37:e77\u0026ndash;e77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkp274\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkp274\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKalogianni DP, Kalligosfyri PM, Kyriakou IK, Christopoulos TK (2018) Advances in microRNA analysis. Anal Bioanal Chem 410:695\u0026ndash;713. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00216-017-0632-z\u003c/span\u003e\u003cspan address=\"10.1007/s00216-017-0632-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi H, Yang L, Zhou X et al (2017) A gold nanoparticle-based colorimetric strategy coupled to duplex-specific nuclease signal amplification for the determination of microRNA. Microchim Acta 184:525\u0026ndash;531. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00604-016-2030-1\u003c/span\u003e\u003cspan address=\"10.1007/s00604-016-2030-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi R-D, Yin B-C, Ye B-C (2016) Ultrasensitive, colorimetric detection of microRNAs based on isothermal exponential amplification reaction-assisted gold nanoparticle amplification. Biosens Bioelectron 86:1011\u0026ndash;1016. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bios.2016.07.042\u003c/span\u003e\u003cspan address=\"10.1016/j.bios.2016.07.042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen M, Wang S, Cai C et al (2016) A simple and sensitive resonance light scattering method based on aggregation of gold nanoparticles for selective detection of microRNA-21. RSC Adv 6:83078\u0026ndash;83083. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C6RA12366J\u003c/span\u003e\u003cspan address=\"10.1039/C6RA12366J\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y-L, Lian Y-M, Wang J-K et al (2021) Highly Sensitive and Specific Mass Spectrometric Platform for miRNA Detection Based on the Multiple-Metal-Nanoparticle Tagging Strategy. Anal Chem 93:5839\u0026ndash;5848. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.1c00065\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.1c00065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X, Chen J, Li B et al (2019) Single particle ICP-MS-based absolute and relative quantification of E. coli O157 16S rRNA using sandwich hybridization capture. Analyst 144:1725\u0026ndash;1730. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C8AN02063A\u003c/span\u003e\u003cspan address=\"10.1039/C8AN02063A\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Han G, Xing Z et al (2014) Multiplex DNA Assay Based on Nanoparticle Probes by Single Particle Inductively Coupled Plasma Mass Spectrometry. Anal Chem 86:3541\u0026ndash;3547. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ac404245z\u003c/span\u003e\u003cspan address=\"10.1021/ac404245z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez Morales S, L\u0026oacute;pez-Portugu\u0026eacute;s C, Fern\u0026aacute;ndez-Sanjurjo M et al (2024) Amplification-Free Strategy for miRNA Quantification in Human Serum Using Single Particle ICP\u0026ndash;MS and Gold Nanoparticles as Labels. Anal Chem. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.analchem.4c01904\u003c/span\u003e\u003cspan address=\"10.1021/acs.analchem.4c01904\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBradley VC, Manard BT, Hendriks L et al (2024) Quantifying platinum binding on protein-functionalized magnetic microparticles using single particle-ICP-TOF-MS. Anal Methods 16:3192\u0026ndash;3201. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D4AY00268G\u003c/span\u003e\u003cspan address=\"10.1039/D4AY00268G\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez de Vega R, Lockwood TE, Paton L et al (2023) Non-target analysis and characterisation of nanoparticles in spirits via single particle ICP-TOF-MS. J Anal Spectrom 38:2656\u0026ndash;2663. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3JA00253E\u003c/span\u003e\u003cspan address=\"10.1039/D3JA00253E\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLockwood TE, Gonzalez de Vega R, Du Z et al (2024) Strategies to enhance figures of merit in ICP-ToF-MS. J Anal Spectrom 39:227\u0026ndash;234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3JA00288H\u003c/span\u003e\u003cspan address=\"10.1039/D3JA00288H\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDegueldre C, Favarger PY, Wold S (2006) Gold colloid analysis by inductively coupled plasma-mass spectrometry in a single particle mode. Anal Chim Acta 555:263\u0026ndash;268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aca.2005.09.021\u003c/span\u003e\u003cspan address=\"10.1016/j.aca.2005.09.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLockwood TE, Gonzalez de Vega R, Clases D (2021) An interactive Python-based data processing platform for single particle and single cell ICP-MS. J Anal Spectrom 36:2536\u0026ndash;2544. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D1JA00297J\u003c/span\u003e\u003cspan address=\"10.1039/D1JA00297J\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLockwood TE, Schlatt L, Clases D (2025) SPCal \u0026ndash; an open source, easy-to-use processing platform for ICP-TOFMS-based single event data. J Anal Spectrom 40:130\u0026ndash;136. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D4JA00241E\u003c/span\u003e\u003cspan address=\"10.1039/D4JA00241E\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Yang S, Ren C et al (2022) Deciphering the roles of miR-16-5p in malignant solid tumors. Biomed Pharmacother 148:112703. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2022.112703\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2022.112703\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKavakiotis I, Alexiou A, Tastsoglou S et al (2022) DIANA-miTED: a microRNA tissue expression database. Nucleic Acids Res 50:D1055\u0026ndash;D1061. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkab733\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkab733\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"miRNA, cell lines, Au/Fe simultaneous detection, time of flight ICP-MS","lastPublishedDoi":"10.21203/rs.3.rs-6279613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6279613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroRNAs (miRNAs) are short single stranded RNA sequences that play an important role in the initiation and progression of cancer. Therefore, the present work tries to establish an analytical platform for the quantitative analysis of this miRNA in cancer cell models without enzymatic amplification reactions. The developed assay is based on a sandwich double-hybridization reaction using a capture oligonucleotide conjugated to magnetic iron oxide microparticles and a detection oligonucleotide conjugated to a 40 nm gold nanoparticle, both particles coated with streptavidin. The optimization of the double-hybridization assay is conducted using inductively coupled plasma in single particle mode with a time of flight analyzer (SP-ICP-ToF-MS) for double detection of Au and Fe within the same event. The developed strategy was directly applied to the quantification of miR-16-5p in cell lysates without amplification reactions. For this aim, the cancer cell line of melanoma (A375) was studied, and two sample preparation strategies have been evaluated. Sequence capturing in extracted RNA provided best results allowing the determination at about 200 pM of miR-16-5p (for 2x10\u003csup\u003e6\u003c/sup\u003e cells). This strategy represents one of the few alternatives to obtain absolute quantification of miRNA in biological samples to permit the direct comparison among cell lines without amplification or transformation reactions of the original sequence.\u003c/p\u003e","manuscriptTitle":"Quantitative Analysis of Intracellular Mirna Content Using Dual Gold and Iron Nanoreporters and Single Particle Icp-Tof-Ms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 17:16:13","doi":"10.21203/rs.3.rs-6279613/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-08T15:14:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T03:13:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-05T07:33:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292034391349995842477306857402803635564","date":"2025-04-05T06:54:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167468866232929072018280320787301340382","date":"2025-04-03T02:05:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T07:39:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-31T01:31:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-31T01:30:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-03-21T17:39:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b25a1b1a-02a7-47f0-87d5-b4b4890c0b14","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:09:22+00:00","versionOfRecord":{"articleIdentity":"rs-6279613","link":"https://doi.org/10.1007/s00604-025-07236-4","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2025-06-02 15:57:24","publishedOnDateReadable":"June 2nd, 2025"},"versionCreatedAt":"2025-04-21 17:16:13","video":"","vorDoi":"10.1007/s00604-025-07236-4","vorDoiUrl":"https://doi.org/10.1007/s00604-025-07236-4","workflowStages":[]},"version":"v1","identity":"rs-6279613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6279613","identity":"rs-6279613","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}