Strategies towards submicron size and high performance magnetic PGMA@Fe3O4@SiO2-COOH microspheres with biological application

The separation of target substances is a signi�cant biological detection procedure, where magnetic microspheres can act as high-performance separation materials. However, challenges are still kept to ful�ll all the requirements. In this study, a type of submicron magnetic poly (glycidyl methacrylate) (PGMA) microsphere was prepared with an in situ coprecipitation method, an electrostatic self-assembly method, and a silica surface coating method. Firstly, the PGMA microspheres were synthesized by a soap-free emulsion polymerization method, and surface charge density determined the coagulation process, further in�uencing the size and monodispersity. Then we found the Superparamagnetism properties of magnetic microspheres could be well controlled by the capping agent sodium citrate (Na 3 Cit), and the superparamagnetic critical size was 10.9 nm. Also, the saturation magnetization was well controlled by the Fe 2+ and Fe 3+ concentration, which was correlated with the nucleation rate of Fe 3 O 4 crystal. Furthermore, we proved that the electrostatic self-assembly was guided by pH, and it was proposed to tightly couple the PGMA-NH 2 microspheres with positive charges and Fe 3 O 4 nanoparticles with negative charges. Finally, the PGMA@Fe 3 O 4 microspheres were coated with SiO 2 , surface modi�ed by carboxyl groups for application. The PGMA@Fe 3 O 4 and carboxyl-modied microspheres exhibited saturated magnetization values of 23.73 and 17.73 emu/g, respectively. These microspheres have been effectively utilized for the extraction of DNA from various sources such as Salmonella typhi, monkeypox virus, and clinical swab samples, suggesting the potential of these microspheres for nucleic acid separation in the biomedical domain.


Introduction
In the biomedical eld, DNA, RNA, proteins, enzymes, and polypeptides are usually regarded as crucial analytical parameters, Consequently, the separation and puri cation of these substances constitute a signi cant method for biological detection, pathological research, and product development.Conventional separation techniques, such as lysis, chromatography, and various forms of liquid or solidphase extraction [1][2][3][4].There are various drawbacks associated with this approach, including reduced e ciency, a complicated procedure, and a lack of autonomous functionality.Furthermore, certain chemicals employed in the extraction process exhibit potential risks to human health.To reduce the separation time and reliance on toxic reagents, nowadays magnetic isolation is applied more extensively, because of its notable attributes of high speci city, sensitivity, and e ciency.Meanwhile, the materials and instruments for this new technology are widely developed.
Biological magnetic isolation typically relies on superparamagnetic polymer microspheres that provide several desirable characteristics, including a high density of functional groups, rapid magnetic response, a large speci c surface area, excellent disperse stability, and biocompatibility [5][6][7].This type of material is mainly divided into core-shell (magnetic components serve as either the core or shell), sandwich, dispersion, and other structures.In recent years there has been a signi cant increase in scholarly interest globally regarding the utilization of various magnetic polymer microspheres for separation purposes.For instance, Ge et al. conducted a study in which they synthesized coordination compounds of M 2+ (M = Ni, Co, Cu, and Zn) to modify polystyrene@Fe 3 O 4 microspheres.These changed microspheres exhibited a dispersion structure and were utilized for the isolation of histidine-tagged proteins.The researchers observed that the modi ed microspheres had notable characteristics such as high adsorption capacity, selectivity, and stability [8].Zandieh et al. used a solvothermal method to prepare submicron Fe 3 O 4 nanoparticles, then in situ coated polydopamine on the surface of particles, nally modi ed with spherical nucleic acids to extract target DNA, these core-shell microspheres exhibited notable extraction e cacy and selectivity [9].However, there are still several shortcomings in the microspheres studied and used now.For core-shell microspheres, it is hard to synthesize Fe 3 O 4 particles with a size larger than 200 nm, thus the bulk size or magnetic contents cannot be controlled in a wide range [10][11][12].To disperse microspheres, it is necessary to combine Fe 3 O 4 nanoparticles with monomers prior to the polymerization procedure.This requires the modi cation of Fe 3 O 4 nanoparticles with oleophilic chemicals, hence imposing limitations on the magnetic components.[13][14][15].To solve the shortcomings, in the 1980s Ugelstad et al developed a new type of core-shell microspheres consisting of polymer microspheres as cores and Fe 3 O 4 nanoparticles as shells, which indicates that the size and monodispersity only depend on polymer microspheres [16,17].Besides, the magnetic contents can be easily controlled up to more than 30% [18].To avoid the magnetic contents being oxidized as well as contact with the detection environment directly, inorganic materials like SiO 2 and TiO 2 are usually used as protective layers on the surface of polymer@Fe 3 O 4 microspheres to form sandwich structures [19,20].
The Fe 3 O 4 shell in the composite microspheres is mainly synthesized by an in situ coprecipitation method, which has the same mechanism as the traditional coprecipitation method, i.e., rstly polymer microspheres were mixed with Fe 2+ and Fe 3+ solutions and then a fast coprecipitation reacted under a special environment with high temperature and pH range of 8~14 [21].This method was widely used and has developed into products like Dynabeads ® [22].However, up to now, most of the studies of in situ coprecipitation magnetic polymer microspheres were based on micron-sized polymer spheres [23][24][25][26], and there are few studies focused on submicron and monodispersed polymer spheres, also the bonding mechanism of Fe 3 O 4 nanoparticles and polymer microspheres has not been well explained.Therefore, it is imperative to conduct thorough investigations on materials with elevated Fe 3 O 4 concentrations and submicron-sized magnetic polymer microspheres.In this study, our target is to synthesize monodispersed and superparamagnetic Fe 3 O 4 @polymer microspheres with a controllable size range of 300~600 nm, also increasing the Fe 3 O 4 contents in the microspheres.The soap-free emulsion method was used to prepare PGMA microspheres as the template, and then the microspheres were modi ed by ethylenediamine (EDA) to x amino groups, nally, the PGMA-NH 2 microspheres were added to Fe 2+ and Fe 3+ solutions, and Fe 3 O 4 contents were generated by an in situ coprecipitation method.We rstly reveal the combination mechanism of Fe PGMA microspheres with different sizes were produced by soap-free emulsion polymerization [27,28].In our method, a certain volume of GMA and 45 mL of H 2 O were rst mixed in a four-neck ask, and argon was purged for 30 min to create an O 2 -free environment.Then the mixture was heated to 70 ℃, and 5 mL KPS solution (2.0 wt.%) was added to the ask as an initiator.Keep the polymerization under mechanical stirring (500 rpm) and re ux condensation for 4.5 h, and the products were centrifuged and washed several times with deionized water, nally dispersed in 50 mL of deionized water.About 1.2 g of PGMA microspheres were dispersed in 36 mL of H 2 O in a four-neck ask and then added 36 mL of EDA to the ask.Heated the solution to 90 ℃ and maintained stirring for 12 h to form the resulting products.The PGMA-NH 2 microspheres were centrifuged and washed several times with deionized water and dispersed in deionized water.

In situ coprecipitation to synthesize PGMA@Fe 3 O 4 microspheres
An amount of 1.0 g PGMA-NH 2 microspheres was mixed with 20 mL of H 2 O in a four-neck ask, then added 50 mL of Fe 3+ and Fe 2+ solution(c(Fe 3+ ): c(Fe 2+ ) = 1.6: 1) to the dispersion liquid, and cooled down to 5 ~ 10 ℃ in an ice bath.The ask was evacuated for 20 min, heated to 75 ℃, and purged argon simultaneously.Added Na 3 Cit to the mixture and then added NH 3 •H 2 O to precipitate the Fe 3+ and Fe 2+ ions.The reaction was retained for 1.5 h, and the composite microsphere products were washed several times by magnetic isolation to remove ammonia.Finally, the PGMA@Fe 3 O 4 microspheres were dispersed in about 50 mL of deionized water.
Brie y, 0.10 g of PGMA@Fe 3 O 4 microspheres were added to a mixed solution including 35 mL of EtOH, 4 mL of deionized water, and 2 mL of ammonia in a four-neck ask.0.1 mL of TEOS was dissolved in 5 mL of EtOH and added to the mixture in the ask dropwise.The hydrolysis reaction was maintained at 30 ℃ for 18 h, then 0.05 mL of APTES was dissolved in 2.5 mL of EtOH and added to the mixture like TEOS.After 6 h, the PGMA@Fe 3 O 4 @SiO 2 -NH 2 microspheres were prepared, washed several times, and dispersed in deionized water.
Weighed 1.5 g of SA to dissolve in 30 mL DMF, the solution was stirred for 4 h and transferred to a fourneck ask, and then 20 mL of DMF was supplied.Took 0.1 g of PGMA@Fe 3 O 4 @SiO 2 -NH 2 microspheres and washed by DMF three times, dispersed in 10 mL of DMF, and added to the SA solution dropwise.Kept the reaction at room temperature, and argon charged several times.The nal products were washed and dispersed in deionized water at a low temperature (~ 4 ℃).

Characterization methods
The morphology of polymer and composite samples was characterized by Zeiss GeminiSEM 360 scanning electron microscope (SEM) and the structure of magnetic microspheres was determined by JEOL JEM-2010 transmission electron microscope (TEM).The crystal structure of Fe 3 O 4 contents was obtained with a Shimadzu XRD-6000 diffractometer equipped with Cu K α radiation.The FT-IR spectra of PGMA and magnetic microspheres were detected by Nicolet iS5 FT-IR spectrophotometer between 4000 and 400 cm − 1 .The magnetic contents and weight loss in high temperature of PGMA@Fe 3 O 4 microspheres were measured by Netzsch STA-449F3 thermal analyzer from room temperature to 800 ℃ at a heating rate of 5 ℃/min under nitrogen.The magnetic hysteresis loops of the samples are measured with a vibrating sample magnetometer (VSM, Lakeshore, Model 7300) with a eld up to 2.0 T.
The dispersion stability in water solutions of magnetic microspheres was measured by standing the dispersion liquid for 30 min, 3 h, and 6 h at room temperature, and the magnetic responsiveness was detected by magnet attraction for 30 s, 1.5 min, and 3 min.

DNA extraction experiment
The biological samples were combined with a lysis solution in order to facilitate the separation of cells or viruses.Subsequently, a substantial amount of polyethylene glycol (PEG) and NaCl were introduced to induce a high concentration of salt and viscosity within the mixture.In this investigation, the solution was augmented by magnetic microspheres, namely PGMA@Fe 3 O 4 @SiO 2 -COOH microspheres synthesized within the scope of this research, or magnetic microsphere products that are commercially accessible.Following that, the microspheres were subjected to the adsorption of DNA molecules onto their surface.A magnetic eld was employed to facilitate the separation process, followed by the removal of the liquid supernatant.Subsequently, pure water was introduced to extract the DNA from the microspheres.The DNA sample was subjected to polymerase chain reaction (PCR) ampli cation to achieve a speci c concentration, and the number of ampli cation cycles was determined.
3 Results and Discussion 3.1 Characterization of PGMA and PGMA@Fe 3 O 4 microspheres As Scheme. 1 illustrated, the core-shell PGMA@Fe 3 O 4 microspheres can be synthesized through an electrostatic self-assembly method.Firstly, the blank PGMA microspheres were prepared through soapfree polymerization and modi ed by EDA.Then, the PGMA@Fe 3 O 4 microspheres with core-shell structure mainly self-assembled with positively charged PGMA and negatively charged Fe 3 O 4 , where EDA and Na 3 Cit act as surfactant modi ers, respectively.Herein, PGMA microspheres are taken as the template to support magnetic Fe 3 O 4 nanoparticles as shell layers on the spherical surface.
During the soap-free polymerization, the acquisition of a well-de ned size distribution of PGMA microspheres is achievable through the surface charge density determined coagulation process.When the GMA volume is 2.25 mL, 4.5 mL, and 6.75 mL (Fig. 1(a)~(c)), the PGMA microspheres, are monodispersed and the average size is about 250 nm, 400 nm, and 600 nm, respectively.However, Fig. 1(d) shows that increasing the GMA volume to 9 mL will result in polydispersity of PGMA microspheres.Such this variation of size and monodispersity of soap-free polymerized PGMA microspheres can be explained by the particle coagulation theory [31][32][33], that is, primary nuclei were formed by precipitation of large chain length polymers, while the ionic initiator provided surface charge to keep dispersity.Under a high GMA concentration, to maintain the thickness of the electrical double layer, the particles coagulated to a larger size to decrease speci c surface area and promoted mono-dispersity by self-sharpening effects.While the GMA concentration was too high, the charge density of primary particles was too low to maintain stability, and the particles coagulate excessively, leading to the polydispersity of nal microspheres.Therefore, the inner mechanism of controlling the size and monodispersity is the adjusting of surface charge density.Meanwhile, maintaining the GMA concentration to synthesize monodispersed PGMA microspheres under a xed initiator amount is signi cant.
To fabricate electrostatically self-assembled PGMA@Fe 3 O 4 microspheres, we prepared positively charged PGMA-NH 2 microspheres, through an EDA triggered a ring-open reaction of epoxy groups on the surface of PGMA microspheres.From the SEM (Fig. 1(e ~ h)) observation, the size and morphology of PGMA-NH 2 microspheres almost appear no changes.For the next step, the PGMA@Fe 3 O 4 microspheres were electrostatically self-assembled through an in situ coprecipitation of Cit 3− capped and negatively charged Fe 3 O 4 nanoparticles on the surface of PGMA-NH 2 microspheres.From the SEM (Fig. 1(i ~ l)) and TEM images (Fig. 1(m ~ p)), the Fe 3 O 4 nanoparticles are mainly distributed on the surface of PGMA microspheres.It makes the whole composite microspheres with rough surfaces and con rms the formation of core-shell structures.There are also some deeply-colored particles in the inner layer, indicating a small part of Fe 3 O 4 nanoparticles are impregnated into PGMA-NH 2 microspheres.

Crystalline and magnetic properties of PGMA@Fe 3 O 4 microspheres
Super-paramagnetism properties are well guaranteed when the grain sizes of Fe 3 O 4 nanocrystals are well controlled below the critical size, i.e., ~ 12 nm.Here we carried out the Na 3 Cit suppressed crystal growth strategy to control Fe 3 O 4 nanocrystals with sizes below ~ 12 nm.In Fig. 2(a), the diffraction peaks match well with standard PDF cards of Fe 3 O 4 crystal, and intensity decreases as the Na 3 Cit amount increases.
The grain size was calculated by the Debye-Scherrer formula.It also decreases with a higher Na 3 Cit amount (see Fig. 2(b)).The essence is concluded by Cornell et al [34]: Cit 3− acts as the capping agent and one Cit 3− ion has a strong coordination effect with one or two Fe ions (Fe 2+ or Fe 3+ ) in the mixture, once ammonia is added to nucleate, the coordinated complex will be distributed on the surface of Fe 3 O 4 nuclei, and Cit 3− produces extra COO − to promote electrostatic repulsion, therefore the growth of Fe 3 O 4 crystal is inhibited [35][36][37].
The addition of Na 3 Cit ≥ 0.050 g is evidenced to get the Fe 3 O 4 grain size below the critical value, thus the coercive force (H c ) and remanent magnetization (M r ) would be meager to exhibit the superparamagnetic behaviors.As the magnetic domain theory described, bulk Fe 3 O 4 has a multidomain structure.While the grain size Fe 3 O 4 decreases, Fe 3 O 4 is transferred to a monodomain structure and the surface spin disorder will be increased [38].Finally, the grain size reaches a critical value, and Fe 3 O 4 materials will have an unstable magnetic state without an external magnetic eld, i.e., superparamagnetism.In Fig. 2(c), it is obvious that the superparamagnetic critical size is at about 10.9 nm, as described in the previous experimental results [39,40].Nevertheless, when the grain size of Fe 3 O 4 is smaller than this value, M r and H c are near zero; when the grain size is more signi cant, the M r and H c increase linearly.From the curves in Fig. 2(d), the saturation magnetization (M s ) is also relative to the Na 3 Cit amount, which is due to the nite size effect and crystal defects [41].In summary, to keep the superparamagnetism and high M s of PGMA@Fe 3 O 4 microspheres, the appropriate Na 3 Cit amount should be 0.050 g.
Besides the superparamagnetism, saturation magnetization is also essential for PGMA@Fe 3 O 4 microspheres, which can be well strengthened by increasing the Fe concentration (c (Fe)).It is correlated with the nucleation rate of Fe 3 O 4 crystal.Figure 3(a) shows that when the ammonia volume added to the mixture is xed, the nucleation process of low and high c(Fe) mixture are different, this result can be interpreted by the classical nucleation-growth theory: A higher concentration will provide a higher degree of supersaturation, thus the absolute value of Gibbs free energy(ΔG) is raised and more Fe 3 O 4 nuclei will form in the initial period [42], after growth, there will be more amount of grains in the distribution system.From Fig. 3(b), (c), and (d), it can be concluded that the M s and Fe 3 O 4 contents are strongly relative to the c(Fe), which is the result of the nucleation process controlled by supersaturation.

The synthesis mechanism of PGMA@Fe 3 O 4 microspheres
A pH-guided electrostatic self-assembly strategy is proposed to tightly couple the PGMA-NH 2 microspheres with positive charges and Fe 3 O 4 microspheres with negative charges.From the curves in Fig. 4(a), the isoelectric point of PGMA-NH 2 microspheres and Fe 3 O 4 @Na 3 Cit nanoparticles are pH = 9.51 and pH = 6.24, respectively.This result indicates that within the pH range of 6.24 to 9.5, both the microspheres and nanoparticles carry opposite surface charges, and the self-assembly process will present.The reason for this phenomenon is that the -NH 2 groups can capture protons from the solution through lone pairs in nitrogen atoms.Thus, the amino groups are protonated, and microspheres carry an extra positive charge in the alkalescent environment [43].Meanwhile, Na 3 Cit acts as the ionic surfactant combined with Fe 3 O 4 nanoparticles and supplies extra negative charge by uncoordinated carboxyl groups in Cit 3− , therefore the isoelectric point is in an acidic atmosphere.It can be clearly seen from Fig. 4(b) that the attraction between -COO − mainly generates the electrostatic self-assembly of a group of Fe 3 O 4 nanoparticles and -NH 3 + group of PGMA microspheres [44].Also, the isoelectric point of PGMA@Fe 3 O 4 microspheres is pH = 8.01, which proves the self-assembly is completed.To make the coprecipitation reaction adequate, and create a strong driving force for self-assembly, we concluded the appropriate synthesis pH range was 8 ~ 9.
To work out the optimum pH range of the electrostatic self-assembly, the introduced ammonia volume and Fe concentration for coprecipitation were stepwise investigated within the pH value of 3 ~ 10.In Fig. 4(c), the reaction system's nal pH increased as more ammonia was added.When V(NH 3 •H 2 O) is 1 mL, the nal pH is only 6.30, where the alkalinity is not high enough to form Fe 3 O 4 , so the conversion rate is low.Increasing V(NH 3 •H 2 O) to 2 mL, the nal pH is 8.51, proving that the coprecipitation reaction is adequate (the suitable pH for coprecipitation is 8 ~ 14), also the Zeta potential of PGMA-NH 2 and Fe 3 O 4 @Na 3 Cit are precisely opposite, thus self-assembly occurred once the Fe 3 O 4 nanoparticles formed.
Continue to increase V(NH 3 •H 2 O) to 4 ~ 6 mL, and the nal pH will be higher than 9.0, where the zeta potential of PGMA-NH 2 microspheres decreases sharply.Therefore, the electrostatic self-assembly will be restrained.In Fig. 4(d), when the V(NH 3 •H 2 O) is xed, the nal pH will decrease as c(Fe) increases, on account of the hydrolysis of Fe 3+ and Fe 2+ producing extra H + .However, the c(Fe) is not the higher, the better, because the initial coating of Fe 3 O 4 nanoparticles will gradually neutralize the positive charge and decrease the Zeta potential of composite microspheres, hence the self-assembly is not continuous.

Biological application of magnetic microspheres
In the above sections, we practically optimized and discussed the synthesis of PGMA@Fe 3 O 4 microspheres through the critical size and pH-guided electrostatic assembly strategies.However, the PGMA@Fe 3 O 4 microspheres tended to clump and precipitate, resulting in a loss of uniform dispersion.
This characteristic renders them unsuitable for meeting the requirements of biological magnetic separation.Whereupon, further modi cation is necessary for the submicron PGMA@Fe 3 O 4 microspheres to get a long-term uniform dispersion.We thus practiced a silica-coating strategy to get a type of carboxyl functionalized microspheres.
After being coated with SiO 2 and modi ed with -COOH, the morphology of magnetic microspheres was well improved to be smoother, and new functional groups could be evidence on the surfaces to resist aggregation.Figure 5(a) and (b) reveal that after SiO 2 coating, the average size of magnetic microspheres increases from 380 nm to 420 nm, and the surface is smoother.We can directly observe from Fig. 5(c) that PGMA@Fe 3 O 4 and PGMA@Fe 3 O 4 @SiO 2 -COOH microspheres are both superparamagnetic and the saturation magnetization is 23.73 and 17.73 emu/g, respectively.The M s of carboxyl microspheres is lower because the SiO 2 coating makes less magnetic content.After functionalized, the microspheres show much higher stabilities in aqueous solutions, because of the raised surface charge density.Figure 5(e) and (f) show that the PGMA@Fe 3 O 4 @SiO 2 -COOH microspheres have higher stability than PGMA@Fe 3 O 4 microspheres and can be well dispersed for at least 30 min.The underlying mechanism might be attributed to the modi cation by the amidation reaction makes microspheres have a higher carboxyl density, thus negative charges on the surface are regained for stable dispersion.The carboxyl microspheres have a relatively high magnetic response within 1.5 min, almost the same as PGMA@Fe 3 O 4 microspheres.Because of the excellent magnetic properties and stability, the PGMA@Fe 3 O 4 @SiO 2 -COOH microspheres synthesized in this study were applied to the separation of DNA in different biological samples, which was mainly based on the cation bridge interaction between the carboxyl group in microspheres and the phosphate group in DNA under a high salt concentration environment [45].
We compared the prepared microspheres with the commercial products (BeaverBeads™ Mag COOH), where the DNA samples from Salmonella typhi, monkeypox virus, and epithelial cells in clinical swabs were detected.As a result, our microspheres show comparable or better performance.To judge the extraction ability, cycle threshold (C T ) is used as the standard index, and the meaning of C T is the times of cyclic ampli cation to reach the detecting concentration [46].As is shown in Table 1, when the microspheres synthesized in our work were used to detect Salmonella typhi and epithelial cell samples, the average C T was lower than the detection using Mag COOH, and in monkeypox virus sample the average C T was only slightly higher, proving the microspheres synthesized our study have the application prospect in isolation of nucleic acid from cells, bacteria, and virus.Based on similar reports recently, in the future, we will connect our research with rare earth uorescent nanoparticles to construct a sensor system [47], and also expand the detection eld to RNA, especially for the detection of RNA in in uenza [48] and COVID-19 virus [48,49].Meanwhile, the detection selectivity of our synthesized carboxyl microspheres under interference conditions will be veri ed for further modi cation.

Conclusions
In summary, we reported a type of submicron monodispersed PGMA@Fe 3 O 4 shell-core microspheres through an in situ coprecipitation and an electrostatic self-assembly method as well as a silica-coated surface modi cation strategy.Through the adsorption of Cit 3− ligands onto the crystalline surface, the growth of Fe 3 O 4 nanocrystals can be well suppressed and their grains can be well controlled below 10.9 nm, the so-called superparamagnetic critical size.This supports the materials to get the remanent magnetization and coercive force as low as near zero.The PGMA@Fe

Figure 5 (
d) indicates that after SiO 2 coating and carboxyl functionalized, the Si-O and Si-O-Si vibration peaks can be observed in turn at 465 and 1078 cm − 1 , and the carboxyl characteristic peak at 1399 cm − 1 is strengthened while stretching vibration peak of C = O bond at 1730 cm − 1 is weakened.However, epoxy groups have obvious vibration peaks at 845 and 910 cm − 1[25] (Fig.S1), proving the ring-open reaction between epoxy and amino groups is complete. Figures

Table 1
The average C T of Mag COOH and PGMA@Fe 3 O 4 @SiO 2 -COOH microspheres in isolation of Salmonella typhi DNA, monkeypox virus, and epithelial cell