# Direct Zeptomole Detection of RNA Biomarkers by Ultrabright Fluorescent Nanoparticles on Magnetic Beads

Elisabete Cruz da Silva, Paraskevi Gaki, Fabien Flieg, Melanie Messmer, Floriane Gucciardi, Yevheniia Markovska, Andreas Reisch, Samira Fafi-kremer, Sébastien Pfeffer, Andrey S Klymchenko

► To cite this version:

Elisabete Cruz da Silva, Paraskevi Gaki, Fabien Flieg, Melanie Messmer, Floriane Gucciardi, et al. Direct Zeptomole Detection of RNA Biomarkers by Ultrabright Fluorescent Nanoparticles on Magnetic Beads. Small, 2024, 20 (45), 10.1002/smll.202404167. [https://doi.org/10.1002/smll.202404167](https://doi.org/10.1002/smll.202404167)

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Submitted on 13 Nov 2024

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# Direct Zeptomole Detection of RNA Biomarkers by Ultrabright Fluorescent Nanoparticles on Magnetic Beads

Elisabete Cruz Da Silva, Paraskevi Gaki, Fabien Flieg, Melanie Messmer, Floriane Gucciardi, Yevheniia Markovska, Andreas Reisch, Samira Fafi-Kremer, Sébastien Pfeffer, and Andrey S. Klymchenko*

Nucleic acids are important biomarkers in cancer and viral diseases. However, their ultralow concentration in biological/clinical samples makes direct target detection challenging, because it leads to slow hybridization kinetics with the probe and its insufficient signal-to-noise ratio. Therefore, RNA target detection is done by molecular (target) amplification, notably by RT-PCR, which is a tedious multistep method that includes nucleic acid extraction and reverse transcription. Here, a direct method based on ultrabright dye-loaded polymeric nanoparticles in a sandwich-like hybridization assay with magnetic beads is reported. The ultrabright DNA-functionalized nanoparticle, equivalent to ≈10 000 strongly emissive rhodamine dyes, is hybridized with the magnetic bead to the RNA target, providing the signal amplification for the detection. This concept (magneto-fluorescent sandwich) enables high-throughput detection of DNA and RNA sequences of varied lengths from 48 to 1362 nt with the limit of detection down to 0.3 fM using a plate reader (15 zeptomoles), among the best reported for optical sandwich assays. Moreover, it allows semi-quantitative detection of SARS-CoV-2 viral RNA directly in clinical samples without a dedicated RNA extraction step. The developed technology, combining ultrabright nanoparticles with magnetic beads, addresses fundamental challenges in RNA detection; it is expected to accelerate molecular diagnostics of diseases.

readily identified by unique viral RNA sequences. Thus, the detection of RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main approach for diagnosis in the COVID-19 pandemic, which uncovered the critical need for rapid, highly sensitive and specific detection methods,[3,4] including those based on nanotechnology.[5-7] RNA biomarkers are generally present at low concentrations in biological samples, frequently below the pM range, and also present complex secondary structures due to intramolecular folding. Therefore, in clinical diagnosis, including that of COVID-19, RNA is detected mainly through a molecular amplification method accompanied by the creation of complementary DNA, which is based on RT-PCR.[3,4,8] However, this gold-standard method requires RNA extraction and purification, multi-step enzymatic reactions, including reverse transcription, and further cyclic amplification, which is prone to false-positive results. Such procedures need skilled personnel, specific equipment,

and long processing times (>2 h), which is difficult to adapt for point-of-care diagnostics. Moreover, the high demand for commercial RNA extraction kits led to a shortage of these reagents, and thus several diagnostic workflows overpassing the long intermediate RNA extraction step were developed.[9-11] In this respect, the possibility of direct detection of RNA in

## 1. Introduction

The ubiquitous nature of RNA makes it an attractive marker for early-stage diagnosis of diseases.[1,2] Aberrant expression of mRNA and non-coding RNA markers can be a signature of pathologies, especially cancer, while viral infections can be

E. Cruz Da Silva, P. Gaki, A. Reisch, A. S. Klymchenko
Laboratoire de Bioimagerie et Pathologies
Faculté de Pharmacie
Université de Strasbourg
UMR 7021 CNRS, Illkirch 67401, France
E-mail: andrey.klymchenko@unistra.fr

E. Cruz Da Silva, P. Gaki, F. Flieg, Y. Markovska
BrightSens Diagnostics SAS
11 Rue de l'Académie, Strasbourg 67000, France
M. Messmer, F. Gucciardi, S. Pfeffer
Architecture et Réactivité de l'ARN
Institut de biologie moléculaire et cellulaire du CNRS
Université de Strasbourg
UPR 9002, Strasbourg 67084, France
S. Fafi-Kremer
CHU de Strasbourg
Laboratoire de Virologie
Université de Strasbourg, INSERM
Strasbourg IRM UMR-S 1109, France

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The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.202404167

© 2024 The Author(s). Small published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

DOI: 10.1002/smll.202404167
biological/clinical samples with minimal processing and without RT-PCR-based amplification is highly attractive, but extremely challenging.[12-15] Isothermal methods of enzymatic target amplification can significantly simplify RNA detection,[16-20] but they still require RNA extraction and reverse transcription steps. On the other hand, the problem of direct enzyme-free methods originates from the background signal and non-specific interactions produced by a multitude of proteins, nucleic acids, and other (bio)molecules.[13] To increase signal-to-noise ratio, the direct detection assays exploit signal amplification, instead of target enzyme-based amplification.[6,21-23] Although the signal amplification assays are less sensitive than the PCR-based target amplification method, they are expected to be quantitative, simpler, and less prone to false positive results.

Fluorescence is among the most popular techniques for molecular detection, featuring high sensitivity, simplicity, and rapidity. It is compatible with a multitude of instrumental techniques, including spectrometers, plate readers, microscopes, and portable devices, including those coupled with smartphones. Nevertheless, fluorescence detection in solution is not suitable below nanomolar concentrations, because the physically limited brightness of organic dyes cannot provide a signal above the background. A number of approaches were explored to enhance the fluorescence signal. One is to amplify the emission of dyes using plasmonic,[24-27] conjugated polymer,[28,29] or dye-loaded polymeric[30-32] nanomaterials. Another solution is to dramatically increase the brightness of the fluorescent emitter using luminescent (fluorescent) nanoparticles (NPs). Indeed, fluorescent NPs attracted attention in the last decades because they can exceed the brightness of organic dyes >100-fold.[33-35] The examples of bright luminescent NPs include semiconductor quantum dots (QDs),[36,37] dye-doped silica nanoparticles,[38] carbon dots,[39,40] and upconversion NPs,[41,42] etc. Among all organic NPs, conjugated polymer NPs,[43,44] aggregation-induced emission NPs,[45,46] and dye-loaded polymeric NPs,[46-48] rapidly gained popularity, because of their high brightness, tunable color, biocompatibility, and rich surface chemistry. We were particularly focused on dye-loaded polymeric NPs because their polymeric matrix allows control of particle size, stable encapsulation of dyes, and robust functionalization with biomolecules, while dye design enables highly efficient emission.[47] Thus, to achieve high particle brightness, we proposed a concept of ionic dye insulation by bulky hydrophobic counterions, which allowed high dye encapsulation with minimized aggregation-caused quenching.[49-51] The concept yielded NPs >3000-fold brighter than organic dyes and ≈100-fold brighter than QDs of similar size.[32,52,53] These NPs showed efficient Förster resonance energy transfer (FRET) to a single dye, generating giant light-harvesting amplification, allowing single-molecule detection at sunlight-like conditions.[30] Moreover, we introduced a charge-controlled nanoprecipitation of hydrophobic polymers bearing few charged groups to achieve the controlled size of polymeric NPs.[53,54] The strategy based on charged amino acids for exposing azide groups at the NP surface allowed further modification with nucleic acids by click chemistry.[31,32] The obtained DNA-NP conjugates yielded ultrabright FRET-based nanoprobes for amplified detection of DNA/RNA in solutions with a picomolar limit of detection,[32] and on surfaces with single-molecule sensitivity,[31] compatible with smartphone-based sensing.[55]

Also, these DNA-functionalized NPs have already been validated for detection of microRNA in cell extracts,[56] and for direct detection of RNA inside cells by RNA-Fluorescence in situ hybridization protocols.[57] However, despite the high brightness and sensitivity of these nanoprobes, they cannot overcome the fundamental problem of slow kinetics of nucleic acid hybridization at ultralow target concentration. Indeed, at the picomolar concentration of the target and probe, the hybridization kinetics were on the time scale of hours, which actually defined the limit of detection of these FRET-based nanoprobes.[31,32,56] The solution is to dramatically increase the concentration of the nanobearing capture sequence, similar to the use of DNA primer excess in PCR. However, it is incompatible with the used FRET-based detection scheme, because the nanoprobe concentration should be close to that of the target.

Therefore, we considered immobilization of target nucleic acid on a solid support, such as a flat glass surface or magnetic bead, followed by further hybridization of the ultrabright NPs, based on the methodology of sandwich assays.[58] In this case, the nanoprobe can be used at high concentrations, while its excess can be removed by simple washing. The sandwich approach with nucleic acids on the glass surface was initially proposed based on gold NPs in combination with silver enhancement, yielding assays with a sensitivity of 50–100 fm for colorimetric assays[59,60] and 20 fm for surface-enhanced Raman spectroscopy (SERS).[61] Using bright fluorescent dye-doped silica NPs in a similar sandwich assay enabled even better sensitivity down to 0.8 fm.[62] However, this method required fluorescence microscopy for NP detection, which is difficult to implement into simple point-of-care or high-throughput detection protocols. Alternatively, one could use iron oxide-based magnetic beads (MBs) to initially capture the target RNA and further hybridize with fluorescent DNA-NPs in order to detect the target directly on the bead. Compared to glass surfaces, MBs have higher surface area and they allow easy manipulation using a magnetic field manually, within microfluidic chips, or in an automated robotic setup.[63,64] They are particularly adapted for the extraction of nucleic acids from biofluids prior to molecular detection.[11,65,66] They can isolate nucleic acids according to surface functionalization.[67] Thus, silica-coated MBs that bind non-selectively to all nucleic acids via electrostatic interactions are used in routine RNA extraction for RT-PCR. On the other hand, magnetic beads functionalized with nucleic acids have been a popular platform for the preparation of sandwich assays for direct RNA/DNA detection. To achieve high sensitivity in these assays, the major efforts were focused on enzymatic amplification of the signal after the sandwich formation and purification, generally using alkaline phosphatase (ALP)[68-70] and horseradish peroxidase (HRP).[70-73] These methods allowed detection by a simple plate reader reaching sensitivities from fm to pm range, with one exception (1.6 am) based on poly-enzyme beads.[73] On the other hand, non-enzymatic sandwich methods that combine MB with luminescent NPs are currently limited to the examples of quantum dots,[74-77] yielding assays with a relatively poor limit of detection, with best performance of 0.5 pm.[74,76] The major problem here is that the conventional detection in solution by a fluorometer or a plate reader is affected by strong light scattering of the micron-sized MBs. We hypothesize that the use of our dye-loaded polymeric NPs, which are much brighter than QDs,[32,52] will allow us to
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Previous A) and the present B) concepts for detection of the nucleic acid target using dye-loaded polymeric NPs. C) The dye R18 with its bulky counterions (F5-TPB) is used for encapsulation inside NPs. Normalized absorption and fluorescence spectra of DNA-functionalized NPs D), their TEM image E), and corresponding particle size distribution histogram F). Scale bar: 50 nm.</figcaption></figure>

overcome the problem of MBs background and enable on-bead detection of RNA with high sensitivity using a standard plate reader and without dedicated RNA extraction protocols.

In the present work, we aimed to develop a method of direct detection of RNA from biofluids for diagnostic applications by the synergy of sequence-specific RNA capture by magnetic beads and on-bead detection with ultrabright fluorescent DNA-NPs. On one hand, sequence-specific hybridization uses oligonucleotide probes complementary to the sequence of interest, which allows target capture on a magnetic bead by hybridization and thus its isolation and enrichment with minimal processing. On the other hand, the second-sequence specific capture will allow direct detection on the magnetic bead surface through binding to our DNA-NPs. This magneto-fluorescent sandwich method enables the detection of target DNA/RNA sequences of different lengths with sensitivity down to 1 fM in 50 µL, that is, 50 zeptomole of the target. As an example, we show application to the detection of SARS-CoV-2 RNA, which was validated on clinical samples from patients.

## 2. Results

In our previously reported FRET-based strategy (Figure 1A), strand displacement of a few acceptors per particle switched the fluorescence of thousands of encapsulated dyes, providing direct signal amplification.[31,32] However, the sensitivity of this method was limited to the low-picomolar range because of too slow hybridization kinetics of the probe with the target caused by: a) the presence of few coding (capture) sequences per particle (molar ratio of coding to non-coding sequence was 1/60)[31] and b) the need to use low concentration of the probe similar to the target (10 pM).[31] Therefore, we propose a totally different strategy, where the target nucleic acid causes the attachment of NPs to a solid support (Figure 1B). In this case, NPs can be coated exclusively

of capture sequences and used at high concentrations (200 pM), because the excess can be washed out. Thus, we increased 60-fold the number of coding capture DNA per particle and the NPs concentration 20-fold with respect to the previous report,[31] which leads to 1200 enhancement in effective DNA capture concentration. In this way, we address the problem of concentration-limited kinetics of hybridization and the sensitivity of the method would rely mainly on the brightness of NPs.

Here, we have chosen our dye-loaded polymeric NPs because of their high brightness, due to the large number of encapsulated dyes R18 with bulky counterion F5-TPB (Figure 1C) that minimizes self-quenching.[49,51] To formulate NPs, we used the recently developed polymer PEMA-AspN3 bearing azide and carboxylate functional groups.[31] The hydrophobic PEMA block in this polymer provides good optical properties for the encapsulated dyes, while carboxylate ensures the formation of small NPs and exposure of the reactive azide group at the NP surface. NPs were formulated by nanoprecipitation of acetonitrile solution of PEMA-AspN3 into phosphate buffer (pH 7.4). R18/F5-TPB dye was loaded inside NPs at 50 wt.% versus polymer (i.e., 33 wt.% with respect to total particle mass) by premixing it with the polymer in acetonitrile before the nanoprecipitation, in line with previously developed methodology.[1] The obtained NPs were 25 nm in size with good polydispersity (Table S1, Supporting Information). Transmission electron microscopy (TEM) confirmed small particle size, that is, 23 nm (Figure 1E; Table S1, Supporting Information). Then, we grafted DNA to the NP surface using a click reaction between a DBCO group of DNA oligonucleotide and an azide of polymeric NPs. The obtained NPs, purified by ultrafiltration, were 32 nm in size according to DLS (Table S2, Supporting Information). The increase by ≈7 nm corresponds to the double thickness of the DNA shell on NPs surface. The absorption and fluorescence spectra of the obtained DNA-NPs corresponded well to encapsulated R18/F5-TPB dye
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Immobilization of DNA-NPs on the glass surface for detection of short nucleic acid target. A) Workflow for sandwich detection of short DNA by direct hybridization of DNA-NPs on immobilized glass surface through biotin-capture. B) Fluorescence images of direct detection of 48-nt DNA target in increasing concentrations (0, 0.1, 1, and 10 pM). 23-nm NPs loaded with 50 wt.% R18/F5-TPB dye (vs polymer) were used. The excitation power density was 0.2 W cm⁻² at 550 nm. Scale bar: 25 µm. C) Quantification of the number of particles in increasing concentrations of DNA target (0, 0.001, 0.01, 0.1, 1, and 10 pM). PBS buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and imaging. The surface area was 315 µm × 315 µm. Error bars are s.d.m. (10 images were analyzed per condition).</figcaption></figure>

(Figure S1, Supporting Information). The fluorescence quantum yield was 63 ± 3% (Table S1, Supporting Information), in line with earlier works on analogous NPs.[31] Given that dye loading is 50 wt% versus polymer and NPs size of 23 nm, the estimated number of dyes per NPs is ≈900. Thus, the brightness of our NPs corresponds to ≈900 dyes with a high quantum yield.

To detect the target, we considered two approaches based on a similar hybridization concept, where the target DNA links NPs to a solid support. In the first approach, the glass surface and NP surface are functionalized with two different capture DNA sequences (Figure 2A). To functionalize the glass surface, we used an established approach based on the absorption of BSA-biotin,[78] further binding of neutravidin (streptavidin analog), and finally the addition of corresponding DNA capture bearing biotin unit (Figure 2A). In the presence of the target DNA/RNA, capture sequences would hybridize with the target, leading to the immobilization of NPs on the glass surface. After a washing step, the latter can be further visualized by fluorescence microscopy. Given the high brightness of these NPs, they could be directly visualized as single particles using a wide-field fluorescence microscope equipped with LED excitation light and a high-sensitivity sCMOS camera, as we showed earlier.[31,32] As a target, we selected a 48-nt N1 fragment of SARS-CoV-2 RNA. One can see that without the target practically no NPs were detected on the glass surface (Figure 2B). Then, the addition of the target led to the appearance of fluorescent dots at the surface and the number of these dots increased clearly with the amount of the target (Figure 2B,C). Based on our previous single-particle studies, using similar experimental conditions,[31,32] the observed dots can be unambiguously assigned to single NPs. Thus, DNA-NPs did not bind non-specifically to the DNA-functionalized glass surface, whereas the target oligonucleotide led to the immobilization of NPs on the glass surface. Importantly, the number of immobilized NPs correlated well with the concentration of the target oligonucleotides (Figure 2C). The limit of detection in these measurements was in the 1–10 fM range, which is remarkably low. One should note that the concentration of NPs used (200 pM) was much larger than the DNA target concentration (1 fM to 10 pM), which excluded the possibility of multiple targets hybridizing with a single nanoparticle. Therefore, we can consider

that a single immobilized nanoparticle corresponds to hybridization of a single target oligonucleotide. The experiments with a negative control DNA target gave a low signal, indicating that the detection is sequence-specific (Figure S2, Supporting Information). These experiments provide the proof-of-concept for nucleic acid detection by the target-driven DNA-NPs immobilization strategy.

Inspired by the results obtained on the glass surface, we aimed to simplify the detection scheme, where one would not need a fluorescence microscope for DNA/RNA detection. Therefore, we replaced the glass surface with magnetic beads functionalized with streptavidin (MBs). The idea to use magnetic beads has two important features. First, magnetic beads are well-established for RNA extraction, so combining them with our NPs could yield a method that would not require a dedicated RNA extraction protocol. Second, magnetic beads are easy to handle, allowing efficient washing and further use in simple detection schemes, for example with the help of a plate reader. In our design of the assay, the target oligonucleotide is hybridized with capture DNA-biotin (the same as in the case of the glass-based approach) used in excess with respect to the target (20–50 nM) and further immobilized on the surface of MBs (Figure 3A). After washing the MBs with the help of a magnetic stand, they were resuspended and mixed with DNA-NPs. In this case, the presence of the target is expected to immobilize DNA-NPs on the bead surface, while the excess of non-reacted DNA-NPs can be washed away using magnetic separation. As for the glass surface method, we used a large excess of DNA-NPs (200 pM) with respect to the DNA target (1 to 10 pM), which excluded the hybridization of multiple targets (or multiple MBs) per nanoparticle. As a result, we will obtain MBs hybridized through the target with fluorescent NPs. This complex could be resuspended and further detected by a fluorescence plate reader, avoiding the use of a fluorescence microscope. Our results show that increasing the concentration of the target led to an augmentation in the fluorescence signal recorded by the plate reader (Figure 3B). Without the target, the signal was very low, close to that for MBs only. Thus, without the target, DNA-NPs do not interact with the beads, while the presence of the target triggers the hybridization of DNA-NPs on the bead surface. The signal was already clearly observed at 1 fM of the target, while the estimated
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Magnetic beads method for detection of a short nucleic acid target. A) Workflow for sandwich detection of short DNA by direct hybridization of DNA-NPs with suspended magnetic beads through biotin-capture. B) Quantification of fluorescence intensity in increasing concentrations of 48-nt DNA target (0, 0.001, 0.01, 0.1, 1, and 10 pM). PBS buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. 23-nm NPs loaded with 50 wt.% R18/F5-TPB dye (vs polymer) were used. Error bars are s.d.m. (n = 3).</figcaption></figure>

limit of detection, based on 3 standard deviations of the blank, was 0.3 fm. Given that the detection volume was 50 µL, the detection limit was 15 zeptomoles in the absolute amount of the target. The obtained results are in line with those obtained with immobilization on glass, suggesting that target-triggered immobilization of DNA-NPs is a robust methodology for nucleic acid detection. However, in the case of magnetic beads, we obtained comparable or even better sensitivity using a simpler and much less expensive fluorescence instrument. To verify the sequence specificity of the oligonucleotide detection, we performed the same bead-based experiments, in the presence of the total RNA lysate from cells, which contains a large variety of RNA sequences (Figure S3, Supporting Information). Importantly, a similar dose-dependent response was observed in the presence of total RNA extracted from cell lysate, which implies that numerous RNA sequences present in the lysate did not interfere with the hybridization processes. These results confirmed that the detection of the nucleic acid target is sequence-specific. The obtained limit of detection was 1 fm, which was close to that without lysate.

Viral RNA is a long molecule, even if in biological or clinical samples the viral RNAs are also present in the form of smaller fragments caused by degradation of relatively unstable RNA molecules. Therefore, we studied longer RNA sequences produced by in vitro transcription (Figure S4, Supporting Information). The first target was 163 mer (163 nt), where the sequence complementary to our biotin-capture DNA is located inside the sequence, while that complementary to the DNA-NPs was at its extremity. The second target was 1362 mer (1362 nt), where both capture sequences were located inside the total target sequence (Figure 4C). They were tested using both glass surface and magnetic bead-based approaches. We found that the 163 nt RNA target could be readily detected by both methods (Figure 4A,B; Figure S5, Supporting Information), showing clear dose-dependency in the particle number (glass surface method) or fluorescence intensity (MBs method). However, it can be noted that the particle number and the fluorescence intensity dropped in both cases compared to 40 nt RNA target. Given the target length is not so different, this significant decrease in the performance of these two techniques is probably linked to the fact that the sequence complementary to the biotin-capture is located in-

side the total sequence of 163 nt, posing steric hindrance issues for the interaction with the neutravidin on the glass surface or the streptavidin on the MBs. On the other hand, the 1362 nt RNA target did not show a clear response, even though some increase in the signal was observed for samples with the target compared to the control. Therefore, we concluded that the combination of long targets with the presence of internal sequences complementary to DNA-NPs decreases the performance of our hybridization approach, independently of the nature of the immobilization surface (glass or MBs). We can speculate that, in addition to steric issues on the glass or MBs surface, it is sterically difficult for DNA-NPs to hybridize with this type of target because it would require an unfavorable and sterically hindered turn in the RNA strand (Figure 4D). Therefore, in the next steps, we made a series of optimizations for both hybridization mechanisms and particles themselves, while focusing on a simpler method based on MBs.

First, to improve the hybridization of NPs with the target sequence located inside the total sequence, we designed a “post-it” sequence, with one end complementary to the target and another end complementary to our DNA-NPs, connected by an A20 linker (Figure 5A). In this case, the steric problem with DNA-NPs could be solved. To validate the method, we tested first this approach on our model 40 nt target and found that the signal showed a clear dose-dependent response (Figure 5B).

Second, to increase the signal obtained within this assay we varied the number of encapsulated dyes per particle. At first, we increased the size of NPs by nanoprecipitation of the same polymer at higher concentrations of salt. The increase in the sodium chloride concentration resulted in an increase in the particle size, in line with the previous work.[53] In the presence of 50 mM NaCl in phosphate buffer, we obtained NPs of 66 nm polymer core according to DLS (Table S1, Supporting Information) and 51 nm according to TEM (Figure S6, Supporting Information). The size by TEM corresponds to ≈10 000 dyes per particle. The absorption and fluorescence spectra of these NPs were similar to those of smaller NPs (Figure S1, Supporting Information). Taking into account that these NPs exhibited good fluorescence quantum yield (58%, Table S1, Supporting Information), they were expected to be ≈10-times brighter than the original smaller NPs. We immobilized DNA-NPs on the glass surface and imaged at the
<figure><img src="image_7.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_10.png" /></figure>

**Figure 4.** Detection of RNA targets of different lengths. A) Quantification of a number of particles in the detection of increased concentrations of 163 nucleotides RNA target or 1362 nucleotides RNA target (0, 1, 10, 100, and 1000 pm) by the immobilized glass surface. Error bars are s.d.m. (10 images were analyzed per condition). The surface area was 315 µm × 315 µm. B) Quantification of fluorescence intensity in the detection of spiked increased concentrations of 163 nucleotides RNA target or 1362 nucleotides RNA target (0, 1, 10, 100, and 1000 pm) by magnetic beads sandwich detection in 2.5 µg of total RNA extract. RNA secure 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. 23-nm NPs loaded with 50 wt.% R18/F5-TPB dye (vs polymer) were used. Error bars are s.d.m. (n = 3). C) Representation of 163 and 1362 nucleotides RNA targets with different locations of target sequence complementary to DNA-NPs. D) Possible difficulties of direct hybridization between DNA-NPs and a target sequence located not in the target extremity.

<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /></figure>

**Figure 5.** The new method of detection for indirect hybridization of DNA-NP. A) Workflow for sandwich detection of short DNA by indirect hybridization of DNA-NPs with suspended magnetic beads through post-it capture. Post-it (DNA) is composed of three fragments: complementary to the target (green), complementary to the DNA of NPs (red), and noncoding (gray). The red arrow shows that after DNA-NP hybridization, there is no steric hindrance at the extremity of the target. B) Quantification of fluorescence intensity in the detection of spiked increased concentrations of 40 nucleotides DNA target (0, 0.01, 0.1, 1, and 10 pm) by magnetic beads sandwich detection in 2.5 µg of total RNA extract using either direct hybridization of DNA-NPs or indirect hybridization through post-it. RNA secure 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. 23-nm NPs loaded with 50 wt.% R18/F5-TPB dye (vs polymer) were used. Error bars are s.d.m. (n = 3).
<figure><img src="image_13.png" /><figcaption>**Figure 6.** Influence of NP characteristics in the sensitivity magneto-fluorescent sandwich assay. A) Influence of NP size: quantification of fluorescence intensity in the detection of spiked increased concentrations of 40 nucleotides DNA target (0, 0.1, 1, and 10 pM) by magnetic beads sandwich detection in 2.5 µg of total RNA extract using 20 or 50 nm core sized DNA-NPs in a hybridization through post-it. RNA secure 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. B) Effect of dye loading and NPs concentration on the fluorescence signal recorded in the presence of magnetic beads without the target. 50-nm NPs were used. The excitation wavelength was 535 nm. The data for QDs (QD-605) are shown for comparison. The excitation wavelength was 405 nm (QDs/405) and 535 nm (QDs/535). The same power density was applied for both excitation wavelengths. PBS buffer with 0.01 g L⁻¹ Tween 80 and 0.1 mg of magnetic beads were systematically used for detection. C) Effect of dye loading on the detection of target oligonucleotide. Fluorescence intensity for different 40 nt DNA target concentrations (0, 0.1, 1, and 10 pM) in 2.5 µg of total RNA extract, using DNA-NPs at 0.1, 1, 10, and 50 wt.% R18/F5-TPB dye loading (vs polymer) in indirect hybridization through the post-it. RNA secure 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. Error bars are s.d.m. (n = 3).</figcaption></figure>

single-particle level by epi-fluorescence microscopy. Both 20- and 50-nm NPs appeared as dots in the microscopy images (Figure S7, Supporting Information). Quantitative analysis suggested that the latter were tenfold brighter, which was in agreement with the estimated difference in the number of encapsulated dyes per particle. Next, we tested these brighter NPs using the hybridization method with a post-it DNA and found significant improvement in the signal intensity for all tested 40 nt oligonucleotide target concentrations (Figure 6A). Thus, larger NPs indeed provided a stronger response to the target in all studied concentrations, in line with their higher brightness these NPs.

The performance of the assay is directly linked to the particle brightness, because the latter is the key to overcoming light-scattering background from MBs. To investigate this point, we performed model experiments where we measured the fluorescence intensity of our 50-nm DNA-NPs at different concentrations in the presence of magnetic beads without the target. We varied the dye loading inside NPs between 0.1 and 50 wt.% with respect to the polymer and checked at which concentration of NPs, the signal was above the light-scattering background of MBs. It was clear that at the lowest loading, the signal was the same as the background for all studied NP concentrations between 1 fM and 10 nM. In contrast, using NPs loaded with 1 wt.% of dye, we could detect the signal at ≥ 100 fM NPs. Finally, the best result was observed for 50 wt.% dye-loaded NPs, where the signal was already detectable at ≥ 1 fM of NPs. These results were compared with commercial QD-605 excited at the optimal wavelength of 405 nm and at 535 nm, the same as for NPs. We found that for both excitation wavelengths, the signal was detectable above the background only at ≥ 1 pM, although for 405 nm both the signal and the background were much higher. Thus, QDs-605 requires >1000-fold higher concentration to be detected compared to 50 wt.% loaded NPs (Figure 6B). Two reasons could explain the much better performance of dye-loaded NPs compared to QD-605. First, our NPs at 50 wt.% loading contains ≈10 000 dyes NPs with total molar extinction coefficient ≈10 000 × 10⁵ = 10⁹ M⁻¹ cm⁻¹ with a high quantum yield of 58%,

so that they are expected to be >100-fold brighter than QD-605 even being excited at their optimal wavelength of 405 nm, where their extinction coefficient is 4.4 × 10⁶ M⁻¹cm⁻¹ (data from the provider). Second, the use of excitation at 405 nm, optimal for QDs-605, produced a much stronger background compared to that at 535 nm optimal for dye-loaded NPs. We expect that the excitation in the violet region should result in much higher light-scattering and auto-fluorescence of MBs and plastic plate used, which further decreased the signal-to-noise ratio in the experiments with QD-605. At the longer wavelengths, the background was much lower, but this excitation also produced a much lower signal from QD-605, so the detection of QD-605 remained inefficient. The use of dye-loaded NPs provided a much higher signal and allowed the use of long excitation wavelengths with lower background.

Then, using the same DNA post-it method as above, we observed that NPs with low dye-loading (0.1 and 1 wt.% vs polymer) showed poor signal for target concentrations below 10 pM. In contrast, at dye loadings of 10 and especially 50 wt% versus polymer, the signal could be clearly observed at all tested 40 nt target concentrations, including 0.1 pM (Figure 6C). We can conclude that for the magnetic bead method high brightness of DNA-NPs is of utmost importance: ~100 dyes per NP (1 wt.% dye loading) are not sufficient for good performance of the assay, while good results are observed starting from ≈1000 dyes per NP (10 wt.% loading). At low dye loading, a large number of NPs need to be hybridized on the surface of MBs to obtain a fluorescence signal above the scattering background from MBs. In contrast, for ultrabright NPs, only a few hybridized DNA-NPs are sufficient to detect the signal, so that very low concentrations of the target can be detected. Therefore, in the next experiments, we focused on large (50 nm) NPs at 50 wt.% dye loading.

We also verified the importance of the linker between the biotin and the capture sequence, which could facilitate target capture at the solid surface,[79] and we thus tested A10 and A20 linkers. It was clear that the longer linker showed advantages in terms of lower background noise for control without the target (Figure
<figure><img src="image_14.png" /><figcaption>**Figure 7.** Improved detection method for long RNA target. A) Scheme of the sandwich for detection of long RNA by indirect hybridization of DNA-NPs with suspended magnetic beads through a post-it capture. B) Quantification of fluorescence intensity in the detection of spiked increased concentrations of 163 nucleotides RNA target or 1362 nucleotides RNA target (0, 0.01, 0.1, 1, and 10 pM) by magnetic beads sandwich detection in 2.5 µg of total RNA extract using 50 nm core sized DNA-NPs (50 wt.% R18/F5-TPB dye loading vs polymer) in indirect hybridization through post-it and biotin capture with TEG motif in the 20 adenines linker. RNA secure 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. C) Quantification of fluorescence intensity in the detection of spiked increased concentrations of 1362 nucleotides RNA target (0, 0.1, and 10 pM) by magnetic beads sandwich detection in 0.5 µg of total RNA extract using 20 or 45 nucleotides capture sequences (post-its and biotin-TEG-A20 capture oligonucleotides. PBS 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. Error bars are s.d.m. (n = 3).</figcaption></figure>

S8, Supporting Information), which significantly improved the sensitivity of the method. Finally, our optimized conditions contained the following key improvements: 1) increase in the particle size to 50 nm, while keeping 50 wt.% dye loading; 2) DNA post-it; 3) A20 linker for the biotin-capture DNA.

Using this optimized methodology (Figure 5A,7A), we tested 163 and 1362 nt RNA targets, which were spiked into total RNA extract. In addition, we verified the importance of the tetraethylene glycol (TEG) linker between the oligonucleotide and biotin, which was reported to improve the biotin capture by the beads.[80] In the case of the 163 nt target, an excellent response of our optimized assay was observed, while the presence of a TEG linker improved the signal to a small extent (Figure S9, Supporting Information). The response was much better compared to the original method: the detectable signal was clearly seen for the 10 fM target versus 1 pM for the original method. However, the fluorescence response of the method was far lower in the case of long RNA (1362 nt) compared to 163 nt RNA (Figure 7B) or the short DNA target, despite the use of a TEG linker. We expect that a high extent of secondary folded structure in longer RNA decreases the binding strength of our 20-mer capture sequences. Magnesium, which is often used in hybridization buffers to increase the strength of binding,[81] did not significantly improve the response to the long RNA (Figure S10, Supporting Information). Higher temperatures can be used to destabilize RNA secondary structures.[82] We performed RNA secondary structure prediction of the 1362 nt target at different temperatures, from 40 °C (actual temperature used for hybridization) to 75 °C (Figure S10 and Table S3, Supporting Information). At lower temperatures (40 °C), a stable folded structure was suggested by the observed low minimum free energy (MFE) (Table S3, Supporting Information). At higher temperatures (65 °C), we found higher values of entropy of predicted RNA structure, implying that the RNA presents more unfolded areas (Figure S11, Supporting Information). To increase the hybridization temperature, we increased the capture sequences from 20 to 45 nt length, passing from a melting temperature of ≈60 to ≈75 °C. We designed different capture sequences spaced all over

the full length of the 1362 nt RNA target and tested them in different combinations with optimized concentrations (Figure S12, Supporting Information). Then, we tested different hybridization temperatures (Figure S13A, Supporting Information), and chose to use 65 °C for the next experiments. When comparing 20 and 45-nucleotide length capture sequences, we found that longer captures drastically improved the sensitivity of the assay to 1362 nt RNA (Figure 7C). This increase in sensitivity can be explained by a high capture efficiency associated with a stronger hybridization between 45 nucleotide capture sequences and the 1362 nt RNA target. Moreover, we further optimized the hybridization time, while keeping a total assay time of 140 min (Figure S13B, Supporting Information) and increased the amount of magnetic beads, which allowed us to improve the assay sensitivity (Figure S14, Supporting Information). In addition to optimization of hybridization conditions, we adapted the protocol to be entirely performed within a standard 96-well plate, using a commercially available magnet stand, which enables a high-throughput operation with 96 samples simultaneously, in contrast to the originally used magnetic stand for 12 centrifugal tubes.

To validate all series of improvements mentioned above, we performed LOD titration curves. With our optimized method, we could readily detect 1 fM of 1362 nt RNA target, with a signal statistically different from the background (Figure 8A). In parallel, different dilutions of the 1362 nt RNA target were evaluated by the golden standard method for RNA detection, RT-PCR (Figure 8B). Remarkably, our LOD of 1 fM for 1362 nt RNA corresponded to a value of 32 cycles of RT-PCR (Figure 8B). So, the improved method allowed the detection of long RNA spiked within RNA extract with fM sensitivity. These results provided a robust proof of concept for viral RNA detection by a new method combining ultrabright DNA-NPs with MBs capture.

The next challenge was to detect endogenous RNA from SARS-CoV-2 infected cells. The SARS-CoV-2 RNA is long (30 000 nucleotides) and highly folded.[83] First, we tested the response of our optimized assay for the detection of SARS-CoV-2 RNA in RNA extracts from SARS-CoV-2 infected cells. Using
A

<figure><img src="image_15.png" /></figure>

B

<figure><img src="image_16.png" /></figure>

C

<figure><img src="image_17.png" /><figcaption>**Figure 8.** Limit of detection of optimized RNA detection method using ultrabright NPs in comparison to RT-PCR. A) Quantification of fluorescence intensity in the detection of spiked increased concentrations of 1362 nt RNA target in 0.5 µg of total RNA extract using our optimized detection method with magnetic beads. PBS 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. \* *p* < 0.05, \*\*\*\*p < 0.0001. B) RT-PCR data for the same solutions of diluted 1362 nt RNA target: the value of Ct versus the target concentration. C) Endogenous detection of RNA using ultrabright NPs detection method. Quantification of fluorescence intensity in the detection of full-length SARS-CoV-2 using 1 µg of total RNA extract from infected cells. PBS 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. \*\* *p* < 0.001, \*\*\*\*p < 0.0001. Error bars are s.d.m. (n = 3).</figcaption></figure>

1 µg of total RNA, we performed parallel detection using our methodology and RT-PCR. Compared to the RNA extract of non-infected cells, our method showed a statistically significant increase in the signal from the infected samples corresponding to a Ct 32 (Figure 8C). Importantly, with the gradual decrease in the Ct values by RT-PCR of the infected sample, corresponding to more concentrated viral RNA, we observed a gradual increase in the signal by our method (Figure 8C). Thus, our optimized magneto-fluorescent sandwich method provides quantitative information about the level of SARS-CoV-2 RNA in the extracts of the infected cells, correlating with RT-PCR data, which is realized without any enzymatic amplification of the target.

Ultimately, we attempted to detect SARS-CoV-2 RNA directly from clinical samples without RNA extraction. We hypothesized that magnetic beads, which are commonly used for RNA extraction, could effectively capture viral RNA from the clinical samples with the help of biotin-capture DNAs. Moreover, our protocol includes magnetic separation with washing before the application of DNA-NPs, which ensures that the buffer used in the original clinical sample would be removed and thus would not harm our DNA-NPs. For instance, the viral transport media contains inhibitors that reduce the sensitivity of many RT-PCR and RT-LAMP assays.[84,85] SARS-CoV-2 positive clinical samples from the university hospital (CHU) were selected to represent a broad range of Ct values from 18 to 30 by RT-PCR and tested using our optimized NP-based assay (Figure 9). Negative clinical samples were also tested. According to our method, all SARS-CoV-2 positive samples tested showed higher signals compared to the negative ones (Figure 9). The thaw and freeze cycles did not affect our detection (data not shown). Importantly, the signal obtained by our method correlated well with the Ct values obtained by RT-PCR (Figure 9). These results validated the capacity of our magneto-fluorescent sandwich method to detect quantitatively SARS-CoV-2 directly in the clinical samples without dedicated RNA extraction and amplification steps. The achieved sensitivity in clinical samples is close to 30 Ct of RT-PCR, which is in line with the data obtained on model 1362 nt RNA target fragment and on the full-length viral RNA from the cell extracts.

### 3. Discussion

There is a vast variety of methods for RNA detection. However, so far molecular diagnostics relies mainly on PCR-based methods. The reasons are multiple, but one should mention the lack of sensitivity and simplicity of newly developed methods.

<figure><img src="image_18.png" /></figure>

B

<figure><img src="image_19.png" /><figcaption>**Figure 9.** Detection of SARS-CoV-2 RNA from clinical samples without RNA extraction. A) Simplified scheme of a workflow for clinical sample testing. B) Quantification of fluorescence intensity in clinical samples characterized by different ranges of Ct values. Samples A1 and B1 are negative. Samples A2 and B2 have a Ct range of 28–30, samples A3 and B3: 24–27 and samples A4 and B4: 20–23. PBS 1x buffer with 0.01 g L⁻¹ Tween 80 was systematically used for incubation and detection. \*\*\*\*p < 0.0001. Error bars are s.d.m. (n = 3).</figcaption></figure>
<table><caption>**Table 1.** Comparison of sandwich-based methods with optical detection modality.</caption><thead><tr><th>Support</th><th>Signal amplification</th><th>Target</th><th>LoD</th><th>Ref.</th></tr></thead><tbody><tr><td>Glass surface</td><td>Gold NPs, silver enhancement</td><td>27-nt DNA</td><td>50 fM</td><td>[59]</td></tr><tr><td>Glass surface (microarray)</td><td>Gold NPs, silver enhancement</td><td>DNA, PCR fragments</td><td>100 fM</td><td>[60]</td></tr><tr><td>Glass surface</td><td>Dye-loaded silica NPs/microscope</td><td>27-nt DNA</td><td>0.8 fM</td><td>[62]</td></tr><tr><td>Glass surface (microarray)</td><td>Dye-loaded silica NPs/microscope</td><td>50-nt DNA</td><td>1.0 pM</td><td>[98]</td></tr><tr><td>Glass surface (microfluidics)</td><td>Dye-loaded liposomes/microscope</td><td>DNA, NASBA amplicons</td><td>0.4 pM/5 fmol</td><td>[99]</td></tr><tr><td>Glass surface</td><td>Gold NPs, silver enhancement/SERS</td><td>30-nt DNA</td><td>20 fM</td><td>[61]</td></tr><tr><td>Polystyrene surface (microplate)</td><td>HRP enzyme</td><td>35-nt DNA</td><td>3.0 pM</td><td>[100]</td></tr><tr><td>Polystyrene capture beads</td><td>ALP enzyme</td><td>viral RNA</td><td>10 amol</td><td>[101]</td></tr><tr><td>Magentic nanoparticles</td><td>bio-bar-code DNA</td><td>48-nt DNA</td><td>1.0 pM</td><td>[102]</td></tr><tr><td>Magentic nanoparticles</td><td>HRP enzyme on bead</td><td>33-nt DNA</td><td>1.6 aM</td><td>[73]</td></tr><tr><td>Magentic nanoparticles</td><td>SERS tags (NAEBs)</td><td>microRNA</td><td>1.0 pM</td><td>[103]</td></tr><tr><td>Magnetic bead</td><td>ALP enzyme</td><td>yeast RNA</td><td>2 fmol</td><td>[68]</td></tr><tr><td>Magnetic bead</td><td>ALP enzyme</td><td>16S RNA of bacteria</td><td>1.7 amol</td><td>[69]</td></tr><tr><td>Magnetic bead</td><td>HRP/ALP enzymes</td><td>33-nt DNA</td><td>5 amol/50 fM</td><td>[70]</td></tr><tr><td>Magnetic bead</td><td>HRP enzyme</td><td>31mer DNA</td><td>10 amol/0.1 pM</td><td>[71]</td></tr><tr><td>Magnetic bead</td><td>HRP enzyme on gold NP</td><td>46-nt DNA</td><td>1 fmol/10 pM</td><td>[104]</td></tr><tr><td>Magnetic bead</td><td>HRP enzyme on gold NP</td><td>46-nt DNA</td><td>1.0 pM</td><td>[72]</td></tr><tr><td>Magnetic bead</td><td>Isothermal HCR amplification</td><td>30mer DNA</td><td>5 fM</td><td>[87]</td></tr><tr><td>Magnetic bead</td><td>Rolling cyclic amplification</td><td>microRNA</td><td>10.0 pM</td><td>[105]</td></tr><tr><td>Magnetic bead</td><td>No amplification, dyes only</td><td>50-nt DNA</td><td>0.1 nM</td><td>[86]</td></tr><tr><td>Magnetic bead</td><td>Dyes only/microscope</td><td>5-7 kbp dsDNA</td><td>5.0 pM</td><td>[106]</td></tr><tr><td>Magnetic bead</td><td>Quantum dots/flow cytometry</td><td>25-100-nt DNA</td><td>3 fmol/3 nM</td><td>[74]</td></tr><tr><td>Magnetic bead</td><td>Quantum dots</td><td>21-30-nt DNA</td><td>20 pM</td><td>[77]</td></tr><tr><td>Magnetic bead</td><td>Quantum dots</td><td>30-nt DNA</td><td>20 fmol/0.5 pM</td><td>[76]</td></tr><tr><td>Magnetic bead</td><td>Dye-loaded polymeric NPs</td><td>40-nt RNA</td><td>15 zeptomol/ 0.3 fM</td><td>This work</td></tr><tr><td>Magnetic bead</td><td>Dye-loaded polymeric NPs</td><td>1362-nt RNA/viral RNA</td><td>50 zeptomol/ 1 fM</td><td>This work</td></tr></tbody></table>

Therefore, the search for ultrasensitive RNA detection is now focused on finding new direct (molecular amplification-free) methods. Among them, the sandwich-based assays are particularly attractive, because they allow exposure of the target RNA to high concentrations of complementary primers/capture sequences, followed by sandwich purification by washing away the non-reacted components. **Table 1** summarizes the main reported sandwich assays, which exploited optical detection output. It can be seen that a few examples used detection directly on glass surfaces. However, to achieve high sensitivity (fM) one needs to use fluorescence microscopy, as shown in a previous work[62] and our present study. We achieved a similar fM sensitivity as that reported before,[62] because an analogous hybridization and sensing concept were used. This is a “digital” method that could in principle achieve a single-molecule sensitivity, because one detected particle corresponds to one hybridized target. However, its sensitivity is limited here by the non-specific interactions of NPs with the surface, which would produce a non-zero background in the absence of the nucleic acid target. Moreover, the method exploits a limited surface area of the flat solid support, which limits the hybridization kinetics of the assay. Finally, this method is rather complex as it requires the use of a dedicated imaging system (epi-fluorescence microscope) and image analysis, which would limit its translation into point-of-care testing. Therefore, the use of magnetic beads is particularly

promising here, because they address multiple limitations of the flat surfaces, such as much higher surface area and simple high-throughput detection using a plate reader. Moreover, given that RT-PCR methods use magnetic beads in their automated RNA extraction protocols, the sandwich assays using magnetic beads could be readily automated within rapid and inexpensive RNA detection assays. Therefore, a large variety of sandwich assays based on magnetic beads has been developed, which can be seen in Table 1. In case when the method is based on single fluorescent dyes as reporters without signal amplification, the sensitivity of the assays is relatively poor, in the range of 0.1 nM.[86] The problem in these assays stems from the strong light-scattering produced by MBs and the limited brightness of organic dyes. Therefore, current high-sensitivity sandwich assays using MBs with optical detection mode rely on enzymatic amplification methods, including horseradish peroxidase (HRP)[70–73] and alkaline phosphatase (ALP),[68–70] where a single enzyme of the hybridized sandwich can produce a large number of optically active molecules for colorimetric or fluorescence detection. However, the best examples of these methods reach sensitivity in the order of 50 fM (5 attomoles) when one enzyme is grafted per sandwich. Further improvement in sensitivity required the use of poly-enzymes (HRP) grafted on a bead (LoD = 1.6 aM)[73] or the use of isothermal HCR amplification (LoD = 5 fM).[87]
In order to develop a direct sandwich assay with optical detection without enzymatic amplification, one needs to use ultrabright luminescent nanoparticles. The use of quantum dots, which are significantly brighter than single dyes,[36,37] can dramatically improve the limit of detection compared to single dyes (LoD = 0.5 pM).[76] although it remains poor compared to fM sensitivity of the enzymatic methods above. Our work shows that because of the strong scattering of MBs, QD-605 excited at their optimal violet region (405 nm) can be detected only above 1 pM concentration (Figure 6), which explains the limited sensitivity of the reported method. To address this limitation, we propose for the first time to use dye-loaded nanoparticles in a sandwich assay with MBs. This turned out to be a game changer because 1) our dye-loaded polymeric NPs are much brighter than QDs and 2) they are excited at longer wavelengths (535 nm), which produce a much lower background signal. As a result, these NPs can be detected at fM concentrations despite the background produced by MBs (Figure 6). The resulting RNA/DNA magneto-fluorescent sandwich assay enables the detection of short RNA and DNA at 0.3 fM, which is one of the best performances among optical sandwich assays reported to date, with one exception when poly-enzyme configuration is used[73] (Table 1). It indicates that ultrabright dye-loaded polymeric NPs outperform classical amplification methods based on a single HRP and ALP enzyme per sandwich. Moreover, it is >1000-fold more sensitive than FRET-based detection with similar dye-loaded polymeric NPs based on strand displacement (0.25–5 pM).[31,32] The sensitivity of the latter was limited by the slow kinetics of the strand displacement reaction between the highly diluted target and the nanoprobe. In the present method, we use a much higher concentration of DNA capture sequences: ≈1200-fold higher concentration of DNA capture on DNA-NPs (≈10 nM) compared to the FRET-based nanoprobe,[31,32] which enables sufficiently fast hybridization even at fM concentration of the target.

The other challenge addressed in this work is the detection of long RNA molecules, which stems from two issues: 1) steric hindrance in the formation of a sandwich, especially between two particles, and 2) folding of RNA, which inhibits the hybridization of short complementary oligonucleotides. These problems probably explain why the majority of examples presented in Table 1 deal with short DNA/RNA sequences. Our work showed that DNA-modified NPs have difficulties hybridizing directly on the long strand, which is probably connected with the steric hindrance (Figure 4). This problem was addressed by using of specific “post-it” oligonucleotide, which could expose a sticky end for hybridization with DNA-NPs. This approach is similar to the one used for signal amplification assays based on branched DNA,[88,89] however, so far, its application with NPs has been shown only in one study on gold NPs for the detection of PCR DNA fragments (amplicons).[60] Second, we found that the folding of long RNA sequences prevents the hybridization of simple 20-nt sequences, which are commonly used as primers in PCR. Therefore, we proposed to use much longer 45-nt complementary sequences, which produce a twofold effect: they 1) allow hybridization at higher temperatures, where RNA folding is destabilized and 2) make duplexes with post-it more stable than the internally folded RNA duplexes. This approach is inspired by technologies used for RNA enrichment in NGS analysis, where relatively large capture sequences are used.[90,91] Thus, the com-

bination of long complementary capture sequences together with the post-it configuration enabled the magneto-fluorescent sandwich to work with long and folded RNA with LoD of ≈1 fM. To the best of our knowledge, this represents the highest sensitivity reported to date for amplification-free sandwich assays with optical detection, applied to long RNA (Table 1).

One should note that the developed technology is enzyme-free and, therefore, it is hard to compare it with a large variety of indirect enzyme-based methods that amplify the nucleic acid target, such as RT-PCR,[3,4,88] isothermal amplification methods,[16–20] etc. These techniques allow significantly lower LoD, which is typically ≈1000 copies (≈1.7 aM) of SARS-Cov-2 RNA per mL.[92] However, the key advantages of our direct method are 1) no need for reverse transcription, as the hybridization is done directly with target RNA and 2) detection in patient samples without dedicated RNA extraction. On the other hand, CRISPR-based methods enable the detection of RNA targets and provide signal amplification by cleaving reporter DNA/RNA molecules and, thus, generating a fluorescent or colorimetric readout. Without dedicated molecular pre-amplification of the target, the sensitivity is typically in the range of pM,[93] and therefore this technique typically uses a pre-amplification step.[3,94,95] Some recently improved amplification-free CRISPR-based assays allow reaching low fM detection limit,[96,97] which is comparable to the present enzyme-free method.

## 4. Conclusion

We designed a highly sensitive RNA sensing technology, which is based on ultrabright DNA-functionalized dye-loaded polymeric NPs, magnetic beads, and corresponding primers. We found that target-driven immobilization of DNA-NPs on solid support allows the detection of RNA/DNA targets on the glass surface by fluorescence microscopy and directly on magnetic beads by a fluorescence plate reader. The latter method (so-called magneto-fluorescent sandwich) is of particular interest because it could enable simple and automated high-throughput RNA detection in biological samples. We found that the brightness of NPs plays an essential role in obtaining a good fluorescence signal even for low RNA concentration, allowing LOD down to 0.3 fM (15 zepto-moles in the absolute amount of the target). Here, the signal amplification for the detection is achieved by the ultrabright DNA-NP, equivalent to ≈10 000 strongly emissive rhodamine dyes, which is hybridized with the magnetic bead through the RNA target. Moreover, the detection of long RNA was found challenging, probably because of slower diffusion kinetics and RNA folding that can block target sequence accessibility from the inside of the total sequence. Therefore, it was important to i) implement a DNA post-it as an intermediate between the long RNA and our DNA-NPs, ii) ensure sufficient linker length for the biotin-capture DNA, iii) increase the length of the capture sequences to perform a hybridization at 65 °C, and iv) ensure a high concentration of complementary sequences to minimize the effect of slow diffusion kinetics of long RNA. Our optimized magneto-fluorescent sandwich assay allowed the detection of a synthetic 1362 nt RNA fragment of SARS-CoV-2 with fM sensitivity as well as the detection of full-length viral RNA in extracts of SARS-CoV-2 with sensitivity corresponding to 32 Ct by RT-PCR. Ultimately, it allowed us to detect SARS-CoV-2 RNA in clinical samples with
sensitivity equivalent to 30–32 RT-PCR cycles. Owing to a combination of DNA-NPs with magnetic beads, our method was able to detect SARS-CoV-2 RNA directly in the clinical samples without dedicated RNA extraction and amplification steps. This work presents a new technology for simple and rapid RNA detection, where ultrabright DNA-NPs provide ultimate sensitivity, while the use of magnetic beads allows for bypass RNA extraction and enables the use of simple detection mode by a plate reader. The magneto-fluorescent sandwich method could be readily extendable to other types of RNA, which would allow rapid molecular diagnostics of various diseases, especially viral infections and cancers.

## 5. Experimental Section

**Reagents:** Chemicals were purchased from either Sigma Aldrich, Alfa Aesar, or Thermofisher Scientific.

Polymer PEMA-AspN3-5% was synthesized as described previously.[31]

Rhodamine B octadecyl ester tetrakis (penta-fluorophenyl)borate (R18/F5-TPB) was synthesized by ion exchange and purified by column chromatography as described previously.[49]

The oligonucleotide sequences used in this study are shown below:

<table><thead><tr><th>Name</th><th>Sequence (5'-3')</th></tr></thead><tbody><tr><td>N1 - NP</td><td>GGT CCA CCA AAC GTA ATG CGG GGT AAA AA - (DBCO-TEG)</td></tr><tr><td>N1 - Biotin</td><td>(Biotin-TEG) - AAA AAA AAA AAA AAA AAC ACT GCG TTC TCC ATT CTG GTT</td></tr><tr><td>N1-Biotin 1</td><td>(Biotin-TEG) - AAA AAA AAA AAA AAT CTT CCT TGC CAT GTT GAG TGA GAG CGG TGA ACC AAG ACG CAG TA</td></tr><tr><td>N1-Biotin 2</td><td>(Biotin-TEG) - AAA AAA AAA AAA AAG CGA CTA CGT GAT GAG GAA CGA GAA GAG GCT TGA CTG CCG CCT CT</td></tr><tr><td>N1-Biotin 3</td><td>(Biotin-TEG) - AAA AAA AAA AAA AAG GGG GAC AAA AAA GGG GGA AGG GGG GGC ACG AAG GCT CAT CAT TC</td></tr><tr><td>N1 - SEQ B</td><td>GGT CCA CCA AAC GTA ATG CGG GGT AAA AAA AAA AAA AAA AAA CTG AGT CCA GCT CGA AAC TTA</td></tr><tr><td>N1-P1-SEQ B</td><td>TTG ATC GCG CCC CAC TGC GTT CTC CAT TCT GGT TAC TGC CAG TTG AAA AAA AAA AAA AAA AAA CTG AGT CCA GCT CGA AAC TTA</td></tr><tr><td>N1-P2-SEQ B</td><td>GCC AAT TTG GTC ATC TGG ACT GCT ATT GGT GTT AAT TGG AAC GCC AAA AAA AAA AAA AAA CTG AGT CCA GCT CGA AAC TTA</td></tr><tr><td>N1-P3-SEQ B</td><td>CAA GCT GGT TCA ATC TGT CAA GCA GCA GCA AAG CAA GAG CAG CAT AAA AAA AAA AAA AAA CTG AGT CCA GCT CGA AAC TTA</td></tr><tr><td>N1 - SEQ C</td><td>GGT CCA CCA AAC GTA ATG CGG GGT AAA AAA AAA AAA AAA AAT GTC GAC GAG GTC CGA GTG TAA</td></tr><tr><td>N1 - NS</td><td>GGT CCA CCA AAC GTA ATG CGG GGT AAA AAA AAA AAA AAA AAG TAT CTC TTC ATA GCC TTA T</td></tr><tr><td>N1 - small target</td><td>ACC CCG CAT TAC GTT TGG TGG ACC CTA ACC AGA ATG GAG AAC GCA GTG</td></tr><tr><td>N1 - 163 nt target</td><td>CAA ACU AAA AUG UCU GAU AAU GGA CCC CAA AAU CAG CGA AAU GCA CCC CGC AUU ACG UUU GGU GGA CCC UCA GAU UCA ACU GGC AGU AAC CAG AAU GGA GAA CGC AGU GGG GCG CGA UCA AAA CAA CGU CGG CCC CAA GGU UUA CCC AUU AAU ACU GCG UCU U</td></tr></tbody></table>

<table><tbody><tr><td>N1 - 1362 nt target</td><td>AAU UGU GCG UGG AUG AGG CUG GUU CUA AAU CAC CCA UUC AGU ACA UCG AUA UCG GUA AUU AUA CAG UUU CCU GUU UAC CUU UUA CAA UUA AUU GCC AGG AAC CUA AAU UGG GUA GUC UUG UAG UGC GUU GUU CGU UCU AUG AAG ACU UUU UAG AGU AUC AUG ACG UUC GUG UUG UUU UAG AUU UCA UCU AAA CGA ACA AAC UAA AAU GUC UGA UAA UGG ACC CCA AAA UCA GCG AAA UGC ACC CCG CAU UAC GUU UGG UGG ACC CUC AGA UUC AAC UGG CAG UAA CCA GAA UGG AGA ACG CAG UGG GGC GCG AUC AAA ACA ACG UCG GCC CCA AGG UUU ACC CAA UAA UAC UGC GUC UUG GUU CAC CGC UCU CAC UCA ACA UGG CAA GGA AGA CCU UAA AUU CCC UCG AGG ACA AGG CGU UCC AAU UAA CAC CAA UAG CAG UCC AGA UGA CCA AAU UGG CUA CUA CCG AAG AGC UAC CAG ACG AAU UCG UGG UGG UGA CGG UAA AAU GAA AGA UCU CAG UCC AAG AUG GUA UUU CUA CUA CCU AGG AAC UGG GCC AGA AGC UGG ACU UCC CUA UGG UGC UAA CAA AGA CGG CAU CAU AUG GGU UGC AAC UGA GGG AGC CUU GAA UAC ACC AAA AGA UCA CAU UGG CAC CCG CAA UCC UGC UAA CAA UGC UGC AAU CGU GCU ACA ACU UCC UCA AGG AAC AAC AUU GCC AAA AGG CUU CUA CGC AGA AGG GAG CAG AGG CGG CAG UCA AGC CUC UUC UCG UUC CUC AUC ACG UAG UCG CAA CAG UUC AAG AAA UUC AAC UCC AGG CAG CAG UAG GGG AAC UUC UCC UGC UAG AAU GGC UGG CAA UGG CGG UGA UGC UCU UGC UUU GCU GCU UGA CAG AUU GAA CCA GCU UGA GAG CAA AAU GUC UGG UAA AGG CCA ACA ACA ACA AGG CCA AAC UGU CAC UAA GAA AUC UGC UGC UGA GGC UUC UAA GAA GCC UCG GCA AAA ACG UAC UGC CAC UAA AGC AUA CAA UGU AAC ACA AGC UUU CGG CAG ACG UGG UCC AGA ACA AAC CCA AGG AAA UUU UGG GGA CCA GGA ACU AAU CAG ACA AGG AAC UGA UUA CAA ACA UUG GCC GCA AAU UGC ACA AUU UGC CCC CAG CGC UUC AGC GUU CUU CGG AAU GUC GCG CAU UGG CAU GGA AGU CAC ACC UUC GGG AAC GUG GUU GAC CUA CAC AGG UGC CAU CAA AUU GGA UGA CAA AGA UCC AAA UUU CAA AGA UCA AGU CAU UUU GCU GAA UAA GCA UAU UGA CGC AUA CAA AAC AUU CCC ACC AAC AGA GCC UAA AAA GGA CAA AAA GAA GAA GGC UGA UGA AAC UCA AGC CUU ACC GCA GAG ACA</td></tr><tr><td>SEQ B - NP</td><td>(DBCO-TEG) - AAA AAT AAG TTT CGA GCT GGA CTC AGT</td></tr><tr><td>SEQ C - NP</td><td>(DBCO-TEG) - AAA AAT TAC ACT CGG ACC TCG TCG ACA</td></tr><tr><td>NS - NP</td><td>(DBCO-TEG) - AAA AAA TAA GGC TAT GAA GAG ATA C</td></tr></tbody></table>

**Clinical Samples:** The clinical samples used were collected from the Department of Microbiology of the University Hospital Center of Strasbourg (CHU). In total, six nasopharyngeal swab samples were from patients with qRT-PCR-positive SARS-CoV-2. Two nasopharyngeal swab samples were from qRT-PCR-negative patients. All samples arrived in an inactivating lysis medium. The experiments with the clinical samples, which originate from the anonymized collection, were approved by the ethics committee of Strasbourg University Hospitals “COVID-HUS”, reference CE: 2020–34. Patients and/or their relatives have been informed of the storage and use of their samples in the anonymized collection for research purposes, and that they may exercise their right to object.
**RNA Extraction and RT-qPCR:** Total RNA was extracted from Vero-hTMPRSS2 infected with SARS-CoV-2 for 48 h using TRI-Reagent (Invitrogen, Fisher Scientific) according to the manufacturer's instructions. 1 µg of RNA was reverse transcribed using SuperScript IV Vilo (Invitrogen, #11 756 050) according to the manufacturer's instructions.

Real-time-PCR was performed with SYBR Select Master Mix (ThermoFisher Scientific) at an annealing temperature of 60 °C on a CFX96 touch Real-Time PCR machine (Bio-Rad) using the following primers:

<table><tr><td rowspan="2">GAPDH mRNA</td><td>Forward (5'-3')</td><td>CTTTGGTATCGTGAAGGACT</td></tr><tr><td>Reverse (5'-3')</td><td>CCAGTGAGCTTCCCGTTCAG</td></tr></table>

**In Vitro Transcription:** Primer pairs with the forward one containing the T7 promoter at their 5' extremity were designed to amplify regions of nucleocapsid (N) from the SARS-CoV-2 genome. PCR was carried out to amplify the different fragments using Phusion High Fidelity DNA polymerase (ThermoFisher Scientific) from cDNA of Vero-hTMPRSS2 infected with SARS-CoV-2.

Transcripts were synthesized using T7 RNA polymerase (Invitrogen). Briefly, 25 µL of PCR product were incubated with transcription buffer and T7 polymerase at 37 °C for 4 h. After incubation, 2 µL of DNase I was added, and the reaction was incubated at 37 °C for 30 min to digest DNA. The resulting RNA was then purified by phenol-chloroform extraction and ethanol precipitation.

Primer used for in vitro transcription:

<table><thead><tr><td>Name</td><td>Target gene</td><td>Forward primer sequence (5'-3')</td><td>Reverse primer sequence (5'-3')</td><td>Transcript length</td></tr></thead><tbody><tr><td>N1 163</td><td>N</td><td>TAATACGACTCACTATAGGGGCAAACTAAAATGTCTGATAATGGA</td><td>AAGACGCAGTATTATTCGGTAA</td><td>163 bp</td></tr><tr><td>N1 1362</td><td>N</td><td>TAATACGACTCACTATAGGGGAATTGTGCGTGGATGAGGC</td><td>TGTCTCTGCGGTAAAGGCTTG</td><td>1362 bp</td></tr></tbody></table>

**Preparation of Nanoparticles:** Sodium phosphate monobasic (>99.0%, Sigma-Aldrich) and sodium phosphate dibasic dihydrate (>99.0%, Sigma-Aldrich) were used to prepare 20 mM phosphate buffers. The final pH was adjusted with 0.1 M hydrochloride acid or 0.1 M sodium hydroxide. 50 mM of NaCl (Sigma-Aldrich) was added to the phosphate buffer. Milli-Q water (Millipore) was used in all experiments.

A total of 50 µL of the polymer (PEMA-AspN3-5%) solution in acetonitrile (2 mg mL⁻¹ containing R18/F5 at 50 wt.% relative to the polymer) was added quickly using a micropipette to 450 µL of 20 mM phosphate buffer, pH 7.4 with or without 50 mM NaCl at 21 °C under shaking (Thermomixer comfort, Eppendorf, 1100 rpm). While continuing mixing, 500 µL of 20 mM phosphate buffer, pH 7.4 50 mM NaCl were added. Then, the residues of acetonitrile were evaporated.

**General Protocol for Nanoparticles Functionalization with DNA:** Lyophilized single-strand DNA sequences were purchased from Biomers or Eurogentec, dissolved in Milli-Q water, aliquoted, and stored at -20 °C for further experiments. Aliquots of corresponding DNA-DBCO (concentration of 20 µM in the reaction mixture) were added to 200 µL of corresponding nanoparticles. The reaction was mixed and kept overnight at 40 °C without shaking protected from light. Then the reaction was cooled down to room temperature. The mixture was diluted with 20 mM phosphate buffer to 4 mL and NPs were purified by centrifugation using centrifuge filters (Amicon, 0.5 mL, 100 kD, Sigma-Aldrich) on 1000 g at 20 °C for 5 min. The procedure of centrifugation was repeated 6 times to remove the non-reacted oligonucleotides using the corresponding buffer. The obtained functionalized DNA-NPs were kept in the dark at 4 °C.

**Nanoparticles Characterization:** Dynamic light scattering (DLS) measurements were performed on a Zetasizer Nano ZSP (Malvern Instruments S.A.). The Zetasizer software provided with standard cumulates and size distribution by volume analysis was used to characterize nanoparticles by DLS. For the data analysis, the following parameters were used: for the solvent (water) – temperature 25 °C, refractive index RI 1.33, and viscosity 0.8872 cP. Nanoparticles were assumed to be all homogenous

and spherical in shape. Absorption spectra were recorded on a Cary 5000 scan UV-vis spectrophotometer (Varian). Excitation, emission spectra, and anisotropy were recorded on an FS5 Spectrofluorometer (Edinburg Instruments). For standard recording of fluorescence spectra, the excitation wavelength was set at 530 nm. The fluorescence spectra were corrected for detector response and lamp fluctuations.

**Transmission Electron Microscopy (TEM):** Carbon-coated copper-rhodium electron microscopy grids with a 300 mesh (Euromedex, France) were surface treated with a glow discharge in amylamine atmosphere (0.45 mbar, 5–5.3 mA, 25 s) in an Elmo glow discharge system (Cordouan Technologies, France). Then, 5 µL of the solution of NPs was deposited onto the grids and left for 2 min. The grids were then treated for 1 min with a 2% uranyl acetate solution for staining. They were observed with the Tecnai F20 electron microscope, equipped with a FEG operated at 200 keV. Areas covered with nanoparticles of interest were recorded at 29000× or 50000× magnifications on a GATAN CCD 2K × 2K "US10001" camera. Image analysis was performed using the Fiji software.

**Nucleic Acids Detection on Glass-Surface:** The LabTek chamber (Borosilicate cover glass, eight wells, ThermoFisher Scientific) was pre-treated with 1 M of KOH for 30 min and washed 3 times with PBS followed by incubation with 100 µL of BSA-Biotin (Sigma-Aldrich) 0.5 mg mL⁻¹ in PBS for 20 min. Then, the BSA-biotin solution was removed, and the chamber was washed 3 times with 500 µL of PBS. Next, the chamber was incubated with 100 µL of neutravidin (ThermoFisher Scientific) solution (0.5 mg mL⁻¹ in PBS) for 15 min and washed 3 times with 500 µL of PBS. Then the chamber was incubated with 100 µL of 1 µM solution of biotin-capture sequence in PBS for 20 min and washed 3 times with PBS. Finally, 100 µL of detection solution was deposited with 200 pM of nanoprobes

and different concentrations of the synthetic target in PBS with 0.01 g L⁻¹ of Tween80 and incubated for 1 h at 40 °C in the dark. Before measurements the chamber was washed 3 times and covered with 200 µL of the same buffer.

Single-particle measurements were performed in the epi-fluorescence mode using Nikon Ti-E inverted microscope with a CFI Plan Apo 20× air objective (NA = 0.75). The excitation was provided by light-emitting diodes (SpectraX, Lumencor) at 550 nm. The excitation power density was 0.2 W cm⁻². The exposure time was set to 10 × 500 ms per image frame. The fluorescence signal was recorded with a Hamamatsu Orca Flash 4 sCMOS camera.

Single-particle analysis was performed using the Fiji software. Particle locations were detected through a Fiji routine applied to a projection (maximum intensity) of all obtained frames per experiment. After the automatic background subtraction, the number of particles of interest with a diameter of 15 pixels was then measured. Ten images (1024 pixel × 1024 pixel, 315 µm × 315 µm) per condition were analyzed.

**Nucleic Acids Detection on Magnetic Beads:** Synthetic target solutions at different concentrations in PBS with 0.01 g L⁻¹ of Tween 80 were incubated with 50 or 20 nM biotin-capture sequences and also 2 or 10 nM post-it capture sequences when indicated. Cell lysates and RNA extracts were diluted in PBS to the final volume of 50 µL with 0.01 g L⁻¹ of Tween 80 and incubated with 20 nM of each biotin-capture sequence and 10 nM of each post-it capture sequence as indicated. The solution was heated at 95 °C for 2 min, and incubated at 40 °C for 20 min (for 20 nt capture sequences), or at 65 °C for 40 min (for 45 nt capture sequences) without agitation. Then 0.1 or 0.2 mg of streptavidin magnetic beads, previously washed twice with PBS, were added. The suspension was incubated at 40 °C for 1 h with mild agitation. The complex composed of magnetic beads, target, and capture sequences was separated from non-reacted nucleic acids by magnetic field: MagneSphere Stand 12-hole from Promega for 0.5 mL tubes or NucleoMag SEP from Macherey-Nagel for 96-well plates. The beads were washed thrice with the same incubation buffer. 200 pM of nanoprobes
were added and incubated at 40 °C for 1 h with mild agitation. The beads were washed again thrice with the same incubation buffer, and the complex was resuspended with 50 µL of the same buffer before transferring it to a 96-well black plate. Direct detection of nucleic acid was performed on the bead's surface by measuring intensity fluorescence in the microplate (SPARK, TECAN).

**Single-Particle Fluorescence Microscopy:** Immobilization of DNA-NPs was done as follows. The LabTek chamber (Borosilicate cover glass, eight wells, ThermoFisher Scientific) was washed 3 times with PBS followed by incubation with 100 µL of BSA-Biotin (Sigma-Aldrich) 0.5 mg mL⁻¹ in PBS for 5 min. Then, the BSA-biotin solution was removed, and the chamber was washed 3 times with 500 µL of PBS. In the case of nanoparticle immobilization, the chamber was incubated with 100 µL of neutravidin (ThermoFisher Scientific) solution (0.5 mg mL⁻¹ in PBS) for 5 min and washed 3 times with 500 µL of PBS. Then the chamber was incubated with 100 µL of 1 µM solution of T20-biotin in PBS for 5 min and washed 3 times with PBS. Then nanoprobe solution at 1 µM concentration was deposed and incubated for 1 h at room temperature in the dark. Before measurements the chamber was washed 3 times with PBS with 0.01 g L⁻¹ of Tween80 and covered with 200 µL of the same buffer.

**Single-particle microscopy measurements** were performed in the epifluorescence mode using a Nikon Ti-E inverted microscope with a CFI Plan Apo 60x oil objective (NA = 1.4). The excitation was provided by a light emitting diode (SpectraX, Lumencor) at 550 nm and emission was detected through a 600/50 nm band-pass filter. The exposure time was set to 200 ms per image frame. The fluorescence signal was recorded with a Hamamatsu Orca Flash 4 sCMOS camera.

**Single-particle analysis** was performed using the Fiji software. Particle locations were detected through a Fiji routine applied to a projection (maximum intensity) of all obtained frames per experiment. After the automatic background subtraction, the mean intensities of circular regions of interest with a diameter of 8 pixels around the found particle locations were then measured. At least ten image sequences (512 pixel × 512 pixel) per condition with, on average, 500 particles per sample were analyzed.

**RNA Secondary Structure Prediction:** RNA folding studies were done using a prediction tool (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi), which estimates the backbone traces and minimum free energy (MFE) value differences on the optimal secondary structure of RNA molecule. MFE calculations of nucleotide base pairing, which is represented by a color gradient in the scale of xx. A lower MFE value is representative of a more stable structure. The mountain plot representation of MFE, thermodynamic ensemble (pf), and the centroid structure predictions, show the secondary structures in a height versus position, where the helices are represented in slopes, loops in plateaus, and hairpin loops in the peaks.

**Statistical Analysis:** The data were presented without additional preprocessing. All data are represented as mean standard deviation (± SD), obtained from at least 3 independent experimental setups (n ≥ 3). Statistical analysis between samples was conducted by one-way analysis of variance (ANOVA) followed by a Bonferroni post-test with the GraphPad Prism program (version 5.04) and expressed as a *p*-value. The significance level is controlled by a 95% confidence interval.

## Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

## Acknowledgements

This work was supported by the European Research Council ERC Consolidator Grant BrightSens 648525, ERC Proof of Concept Grant AmpliFISH 899928, SATT Conectus Maturation Grant NanoAntenna, Agence Nationale de la recherche (ANR) Résilience Covid-19 ViRNASens. The authors thank Corinne Crucifix from the FRISBI platform for her help with electron microscopy and BrightSens Diagnostics SAS for fruitful discussions.

## Conflict of Interest

A.S.K. and A.R. are co-founders of BrightSens Diagnostics SAS and E.C.S and P.G. are currently employed by this company. A.S.K., E.C.S., P.G., S.P., and A.R. deposited a patent application on the described technology.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

## Keywords

fluorescent polymeric nanoparticles, magnetic beads, molecular diagnostics, non-enzymatic signal amplification, RNA detection

Received: May 23, 2024

Revised: July 5, 2024

Published online:

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