Optimized and scalable synthesis of magnetic nanoparticles for RNA extraction in response to developing countries' needs in the detection and control of SARS-CoV-2

doi:10.21203/rs.3.rs-34552/v1

Ecuador is one of the most affected countries, with the coronavirus disease 2019 (COVID-19) infection, in Latin America derived from an ongoing economic crisis. One of the most important methods for COVID-19 detection is the use of techniques such as real time RT- PCR based on a previous extraction/purification of RNA procedure from nasopharyngeal cells using functionalized magnetic nanoparticles (MNP). This technique allows the processing of ~10,000 tests per day in private companies and around hundreds per day at local Universities guaranteeing to reach a wide range of the population. However, the main drawback of this method is the need for specialized MNP with a strong negative charge for the viral RNA purification to detect the existence of the SARS-CoV-2 virus. Here we present a simplified low cost method to produce 10 g of nanoparticles in 100 mL of solution that was scaled to one litter by parallelizing the process 10 times in just two days and allowing for the possibility of making ~50,000 COVID-19 tests. This communication helps in reducing the cost of acquiring MNP for diverse biomolecular applications supporting developing country budgets constraints and chemical availability specially during the COVID-19 International Health Emergency.

Laboratory Diagnostics

SARS-CoV-2

COVID-19

Molecular diagnosis

Magnetic nanoparticles

XPS

FTIR

Raman spectroscopy

Ecuador

Developing countries

The prevailing pandemic originated from the unusual coronavirus SARS-CoV–2 that causes the COVID–19 infectious disease has been striking the medical health system world wide due to its rapid propagation person-to-person.^1–4 A prompt and accurate detection method is crucial for the detection of COVID–19 in order to keep its proliferation under control. Recently the genome sequences of SARS-CoV–2 have been fully revealed and thus the use of techniques like real-time, reverse transcription polymerase chain reaction (RT-PCR), has been widely used and developed as kits for the clinical diagnosis of COVID–19. Zhao et al. have shown the possibility of fabricating “simple magnetic nanoparticles” that can be further implemented into the RNA magnetic extraction method that will be used later as template in the real time RT-PCR for the clinical diagnosis of COVID–19.⁵ However, the main drawback of this technique is the need for specialized and homogenous magnetic nanoparticles with a strong negative charge, so that the viral RNA extraction and purification process becomes faster and cleaner improvinging the number of clinical tests per day for COVID–19. Although, even if this technique can be implemented in every country, developing countries like Ecuador and many others in Latin America are struggling to implement it due to the relatively high prices of MNP in the international market and their import time frame. An example of this problem is confirmed by the city of Guayaquil in Ecuador, that has suffered extremely from the results of late detection and control along the explosive expansion of the novel coronavirus.⁶

In this communication, a ‘simplified three step method’ to produce large quantities of MNP for RNA extraction based on Zhao et al. synthesis⁵ is presented, optimized and scaled. This work brings a systematic characterization of the produced MNP along their fabrication process and it can be used as a benchmark in the correct synthesis of homogeneously MNP specialized for a high quality RNA extraction along their potential implementation into a real time RT-PCR for SARS-CoV–2 detection.

Simplified preparation of magnetic nanoparticles

The methodology for the preparation of magnetic nanoparticles coated with a negatively charged polymer (Poly) to be used in the extraction of RNA from COVID–19 is represented in Figure 1a-c. Magnetic nanoparticles were prepared using a simple and low-cost co- precipitation method and coated with (3-Aminopropyl) triethoxysilane (APTES) (Figure 1a). The poly (amino-ester) was prepared based on a modification of the protocol reported by Zhao et al. (Figure 1b). Subsequently, the amino-magnetic nanoparticles (NH₂-MNPs) were coated with the poly (amino-ester) to obtain the negative charge amino-magnetic nanoparticles (Poly-NH₂-MNPs) (Figure 1c). The synthesis protocol reported here was designed to produce 100 mL at (10 % w/v) of Poly-NH₂-MNP. A 100 mL of Poly-NH₂-MNP will permit the execution of about 5,000 RNA extraction/purification procedures from nasopharyngeal cells that will be used as template in the real time RT-PCR tests for COVID- 19 detection. However, the way that this epidemic has been developing around the world requires a more persuasive and robust method that could cover the real needs of developing countries as the amount of people to be tested is much higher every day. Thus, we scaled the synthesis method (Supplementary Fig. 2a) by parallelizing simultaneously each step from Figure 1 being able to increase the production of MNP in one order of magnitude. We are able to obtain 1 L of Poly-NH₂-MNP (10 % w/v) in just two days in a basic laboratory. This result will allow the execution of about 50,000 RT-PCR tests for COVID–19 detection in Ecuador and become a potential solution for developing countries.

Characterization of magnetic nanoparticles as prepared for the RNA extraction method

A morphological analysis of the samples(Figure 1 d-e) was conducted using a scanning electron microscopy (SEM) based on a secondary electrons detector from a Versa Probe III X-ray photoelectron spectrometer. In Figure 1d we confirm that our first treatment for dispersion of MNP was successful and our pristine magnetite nanoparticles were coated. The latter is an important and crucial property of the synthesis of MNP for RNA extraction as the succes in further coatings processes (Figure 1a-c) is determined by an effective initial dispersion and coating of the MNP. The evident cracks along the surface of the dried dispersion of MNP on the SiO₂ wafer confirms no apparent interaction that allows the agglomeration of the nanoparticles as desired. In contrast, for the final end of this synthesis process, one desires to have a continuous and heterogeneous polymer/nanoparticle system that allows the correct performance along the RNA extraction technique. In Figure 1e we corroborate this fact by a noticeable smooth and continuous surface derived from the presence of the poly (amino-ester) coating within the NH₂-MNP. We can identify small agglomeration sites that are the result of a fast drying process (80°C heated) of the drop at the SiO₂ wafer.

In order to prove the efficiency of the coating process described in Figure 1a-c, we employed Raman spectroscopy as a fast and precise characterization tool for nanomaterials. This method is well known in the characterization of magnetic nanoparticles ^7,8 and their interaction with organic solvents, polymers, and molecules.^9,10 In Figure 2, the evolution of the Raman spectrum from the MNP is presented. We can trace the scheme in Figure 1 with respect to the obtained Raman spectra as follows: i) As a first step, the MNP have to be synthesized and dispersed homogeneously in isopropanol solution. We found the characteristic peak from magnetite (Fe₃O₄)^7,11 at 670 cm^–1 using a 633 nm laser excitation and low laser power <1 mW (Figure 2g - red). We also featured the presence of a peak at ~380 cm^–1 resulting from the [OH]^- groups linked to the surface of magnetite nanoparticles (Figure 2c - orange) confirming the initial coating during the first step of the synthesis process as proposed in Figure 1a. ii) The second step of the synthesis requires a special coating with APTES molecules that eclipse the magnetite Raman response by a broad background as observed in Figure 2e. iii) In a third step the APTES treated MNP (NH₂-MNP) are dissolved and washed with DMSO enhancing the magnetite Raman response at 670 cm^–1 due to a slight decoating of the initial APTES shell within the additional emergence of a double peak around 3000 cm^–1 characteristic of DMSO.

iv) Finally, the nanostructured NH₂-MNP is binded to a polymer (amino-ester) that will serve as a negative charge carrier for the optimal RNA extraction methodology. Our Raman measurement of this final sample (Poly-NH₂-MNP) shows again a broad background characteristic of a fluorescent polymer at 532 nm in addition to a perceptible signal of magnetite at 670 cm^–1. During each Raman measurement, optical images of the samples were taken (Figure 2b,d,f,h) to ensure no degradation of the sample during the measurement, and a surface morphological confirmation when compared to Figure 2d-e.

In order to verify the presence of the final nanoparticle coating, that is a crucial step for the effectiveness of the MNP when used in the RNA extraction method, we used two accurate standar techniques: a) Infrared spectroscopy (Figure 3), and b) X-ray photoelectron spectroscopy (XPS - Figure 4).

In Figure 3a the spectrum of magnetite nanoparticles is shown and confirmed by the presence of v(Fe-O) stretching vibration in a tetrahedral site at ~572 cm^–1characteristic of magnetite.^7,13 The distinctive Fe-O-Si band is present around 580 cm^–1 ¹⁴ and overlaps with the magnetite vibration band, however it can be observed that the band in this region considerably increases the intensity, supporting the adsorption of APTES molecules on the magnetite surface (Figure 3b-d) as expected from the first step in Figure 1a.

The particles coated with APTES (NH₂-MNP) show the amino groups in the region from 3200–3400 cm^–1 corresponding to a N-H stretching and at 1630 cm^–1 for the N-H bending vibrations. The characteristic Si-O-Si and Si-OH vibrations are present in the region from 1050–1100 cm^–1.

The infrared measurement of the prepared reference negatively charged polymer coating (Poly) shows a broad band vibration of N-H and OH− groups in the range of 3700–3000 cm⁻¹. The bands at 2860–2920 cm^–1 assigned to C-H stretching symmetric and asymmetric vibrations of the ethylenediamine, the amide bands at 1576 cm^–1 and at, 1515- 1568 cm^–1 to as (COO−) and the other band in the range 1313–1346 cm^–1 assigned to s(COO−), the vibration bands at 1682–1685 cm^–1 (n C = O) of the acrylate group, the 1111 cm^–1 assigned to C-O-C and the adsorption peaks at 1711 cm^–1 characteristic of the C = O stretching corresponding to the carbonyl group.

The infrared spectrum confirms the efficient coating of MNP with Poly. According to Zhao et al. this particular negatively charged coating is essential for the optimal extraction and purification of the RNA sample that will improve the performance of the real time RT-PCR analysis process in the detection of SARS-CoV–2.⁵ Additionally we also have the Fe-O-Fe characteristic band of magnetite at 580 cm^–1 confirming the magnetic behavior also needed during the RNA extraction and purification process.

The final chemical surface characterization for the MNP was conducted using XPS spectroscopy. As it is shown in Figure 4, the binding energy comparative survey analysis exhibit clear features among the samples:

The iron analysis in XPS spectroscopy is a tool to confirm the presence of magnetite (Fe3O4) nanoparticles. Our initial material (Figure 4a) revealed the strongest peak from the Fe2p3 core level with a 24.8 % atomic concentration. By performing a high resolution XPS analysis in the region from 705 to 730 eV, we confirmed the presence of magnetite by a doublet peak corresponding to the Fe2p1/2 and Fe2p3/211 components (Supplementary Fig. 3a). After the first coating process (Figure 4b), we observed an attenuation down to 3.2 (atm %) of the Fe peak derived from the presence of NH4OH deposited on the MNP’s surface. After washing the MNP with DMSO, the signal intensity of Fe2p3 raised to 7.7 (atm %) in Figure 4c as the DMSO washed out the excess of the non-attached NH4OH coating material. In the final step (Figure 4d), the nanoparticles were coated with a polymer (3-Aminopropyl) and thus the signal of magnetite was strongly reduced to less than 0.1 (atm %). In the end, we confirmed that the final coating created on the MNP is of around 9 nm, which is the limit for photon penetration in the sample coming from the XPS surface technique.
Regarding the N1s core level (~400 eV) analysis, we found this peak to be absent (as expected) in the pristine MNP sample in Figure 4a due to the lack of amino groups. After the first coating with NH4OH the nitrogen concentration was increased to 8.4 % (Figure 4b). Subsequently, the excess of NH4OH molecules was washed by DMSO (Figure 4c). We noticed then a reduction in the nitrogen atomic concentration to 7.5 % as is shown in Table 1. We attribute this effect, as observed in the infrared analysis, to the removal of some NH4 groups from the first coating surface of the nanoparticles. In the final step (Figure 4d), when the MNP are being coated with 3-Aminopropyl (Poly-NH2-MNP), the nitrogen concentration is raised to 10.3 atomic % (Table 1). The latter nitrogen development is a clear signature of the successful coating process essential to have magnetic nanoparticles covered with a negative charged polymer for the RNA extraction. The detailed development of the nitrogen functional groups formation on the MNP can be observed in the Supplementary Information in a close region from 398 to 405 eV (Supplementary Fig. 3) showing the characteristic peaks corresponding to NH₄OH and NH₂ molecular elements present in the sample.^15,16
The carbon C1s signal in the pristine MNP (Figure 4a) is minimum (~10 % Table 1) denoting the clean precursors that mostly contain magnetite nanoparticles. The C1s core level appears at (284.8 eV) after the first coating deposition with 41,7 (atm %) in Figure 4b and Table 1 correspondingly followed by a reduction (32.4 %) due to the DMSO processing. At the final part of the process, the C1s signal increased in agreement to the 3-Aminopropyl addition as the last coating (Figure 4d) having a 53.5 (atm %) on the sample.
The O1s feature at (532 eV) for MNP (Figure 4a) has an atomic percentage of 60.4 with a final value of 32.2 % at the final step (see Table 1) confirming an intensity reduction due the signal attenuation of the Fe3O4, leaving just the oxygen contribution from the 3-Aminopropyl coating. Traces of Cl, Si and Na are present at (~196 eV, 101 eV and 1072 eV correspondingly) due the composition of the precursors used in the synthesis and in each consecutive coating process as it is shown in Table 1, where atomic chemical elemental composition for each step is exhibited.

Table 1 \| Atomic percentages of chemical elements from the XPS study.
(Atomic %)	MNP	NH₂-MNP	NH₂-MNP in DMSO	Poly-NH₂-MNP
O1s (%)	60.4	33.9	42.4	32.2
Fe2p3 (%)	24.8	3.2	7.7	<0.1

C1s (%)	10.4	41.7	32.4	53.5
N1s (%)	-	8.4	7.5	10.3
Si2p (%)	-	11	8.7	1.3
Cl2p (%)	4.4	1.8	1.3	0.3
Na1s (%)	-	-	-	2.4

Table 1: Atomic percentages at each coating stage process evaluated from the X-ray photoelectron spectra

In summary, we reported a comprehensive study along the synthesis of functionalized coated MNP with a negatively charged polymer for viral RNA extraction and purification that could be implemented in the detection of the SARS-CoV–2 virus on a large scale. This work set a systematic characterization protocol in the synthesis of MNP that can be used as a benchmark for the correct fabrication of MNP specialized for high quality RNA extraction method before their potential implementation into a real time RT-PCR. The synthesis protocol reported here serves for the production of 100 mL at (10 % w/v) of Poly-NH₂-MNP that can be extended to 1L of Poly-NH₂-MNP (10 % w/v) in just two days in a basic laboratory (Supplementary Fig.

2) by parallelizing the synthesis process allowing for the execution of about 50,000 real time RT-PCR tests for COVID–19 detection. Our final 1L Poly-NH₂-MNP (10 % w/v) was sent to be tested as part of a real time RT-PCR analyses in a private company aiming for the optimal extraction of RNA to detect SARS-CoV–2 virus. With this work we (Supplementary Fig. 4) provided a solution to a national Latinamerican problem along the detection and control of the explosive expansion of the novel coronavirus despite the country budget constraints and chemical and laboratory facility availability during this declared International Public Health Emergency.

Infrared spectroscopy. The infrared analysis of the samples were done using an Agilent Technologies spectrometer Cary 360 with a diamond attenuated total reflectance (ATR) accessory and resolution of 4 cm^–1. For each spectrum 32 scans acquisitions were compiled in a range between 400 and 4000 cm^–1.

Raman spectroscopy. The spectroscopic Raman detection was carried out under ambient conditions using a HORIBA LabRam spectrometer with a 514 nm and 633 nm excitation wavelengths at ~0.65 mW and 1.5 mW between –50 and 4,000 cm^–1. To avoid laser-induced damage on the samples the laser power was kept below 1 mW. Raman measurements were carried out using a micro-Raman setup with a 100X short distance objective in backscattering geometry. A charge-coupled device is used to detect the signal after analysing the signal via a monochromator similar to previous Raman spectroscopy studies we have conducted [¹⁰]. The spectrometer was calibrated in frequency with the Rayleigh peak to be set at 0 cm^–1.

X-ray photoelectron spectroscopy. The surface chemistry characterization was recorded by using X-Ray Photoelectron Spectroscopy (XPS) PHI VersaProbe III (Physical-Electronics) equipped with a 180 hemispherical electron energy analyzer and excited by a monochromatized Al-K_� source with an energy 1486.6 eV. Its energy analyzer operates in the

pass energy mode at 280 eV for Survey and 55 eV for high resolution. The analysis spot had a diameter of 50 μm and 45° detection angle relative to the substrate surface.

Chemicals and solvents:

Iron (III) chloride hexahydrate, Iron (II) chloride tetrahydrate, ammonium hydroxide, (3- Aminopropyl) triethoxysilane (APTES), and dimethyl sulfoxide (DMSO), isopropanol,

ethylenediamine and ethanol, 1,4-butanediol diacrylate and 6-amino caproic acid were bought from Sigma-Aldrich Co. and used as received (99.99 % purity).

Synthesis of Magnetic Nanoparticles (MNP):

Magnetic nanoparticles (MNP) were prepared using a simple and low-cost co-precipitation method (Figure 1a).¹⁷ 6.5 g of Iron (III) chloride hexahydrate and 3.5 g of Iron (II) chloride tetrahydrate were mixed in 100 mL of distilled water at 50 °C, and 20 mL of ammonium hydroxide was added into the mixture with vigorous stirring under a nitrogen atmosphere. A rapid change of solution colour was observed from orange to black, indicating the formation of MNP. The solution mixture was continuously stirred for another 30 min, and the resulting black products (MNP) were collected with a magnet and dispersed into ethanol after washing several times with deionized water and ethanol as in reference [¹⁷].

Synthesis of amino-magnetic nanoparticles (NH₂-MNP):

Subsequently, the prepared MNPs were dispersed into 50 mL isopropanol, and 2 mL of APTES was dropwise mixed with MNPs solution (Figure 1a).¹⁸ The mixture was incubated under continuous sonication for 3 h at room temperature, followed by the collection of amino- modified MNPs (NH₂-MNP) with a magnet and washing with distilled water and ethanol to remove free APTES. The final prepared NH2-MNP were preserved in ethanol and the loading of the -NH₂ group on the surface was verified by infrared spectroscopy (Figure 3) as donde in reference [¹⁸].

Synthesis of polymer coated amino-magnetic nanoparticles (Poly-NH₂-MNP):

Poly-NH₂-MNP synthesis is adapted based on Zhao et al. protocol.⁵ NH₂-MNP were dispersed into 25 mL of 50% (v/v) DMSO aqueous solution and then mixed with 2.5 mL of

Poly (Figure 1c). Subsequently, 2.5 mL of NaOH solution (1 M) was introduced to the mixture, followed by vigorously stirring for 4 h at room temperature in a dark place, followed by the collection of Poly-NH₂-MNP with a magnet and washing it with 50% (v/v) DMSO for several times. The obtained polymer coated MNP (Poly-NH₂-MNP) were stored in distilled water at 4 °C in a dark place as explained in reference [⁵]. The loading of a carbonyl group of Poly (amino-ester) on the surface was verified by infrared spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and SEM techniques (Figures 1 to 4).

Corresponding Author

* E-Mail: [email protected] School of Physical Sciences and Nanotechnology, Urcuquı́ 100119, Ecuador.

* E-Mail:[email protected] School of Physical Sciences and Nanotechnology, Urcuquı́ 100119, Ecuador.

Competing interest

No competing interest

Acknowledgements

The authors thank Frank Alexis and Ernesto Medina for supervising the project as scientific group leaders and principal investigators. The authors thank ONELABT for providing some of the chemicals for these experiments.

Author Contributions

G.G., S.B., J.C.T., D.N., and C.R. provided helpful input for the sample preparation and the optimization of the synthesis protocol for Poly-NH₂-MNP. G.G., S.B., and D.N. synthesized the Poly-NH₂-MNP. J.C.T. and C.R. performed the Raman, SEM and XPS characterization.

G.G. and S.B. conducted the IR characterization and data analysis. J.C.T. conducted the Raman and SEM data analysis. C.R. performed the XPS data analysis. S.B., G.G., and D.N. contributed substantially in the analysis for Poly-NH₂-MNP. J.C.T. wrote the manuscript.

Supplementaryinformation.pdf

Optimized and scalable synthesis of magnetic nanoparticles for RNA extraction in response to developing countries' needs in the detection and control of SARS-CoV-2

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results And Discussion

Simplified preparation of magnetic nanoparticles

Characterization of magnetic nanoparticles as prepared for the RNA extraction method

Conclusions

Methods

Chemicals and solvents:

Synthesis of Magnetic Nanoparticles (MNP):

Synthesis of amino-magnetic nanoparticles (NH₂-MNP):

Synthesis of polymer coated amino-magnetic nanoparticles (Poly-NH₂-MNP):

References

Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Optimized and scalable synthesis of magnetic nanoparticles for RNA extraction in response to developing countries' needs in the detection and control of SARS-CoV-2

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results And Discussion

Simplified preparation of magnetic nanoparticles

Characterization of magnetic nanoparticles as prepared for the RNA extraction method

Conclusions

Methods

Chemicals and solvents:

Synthesis of Magnetic Nanoparticles (MNP):

Synthesis of amino-magnetic nanoparticles (NH2-MNP):

Synthesis of polymer coated amino-magnetic nanoparticles (Poly-NH2-MNP):

References

Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Synthesis of amino-magnetic nanoparticles (NH₂-MNP):

Synthesis of polymer coated amino-magnetic nanoparticles (Poly-NH₂-MNP):