Evaluation of poly(ethylene glycol)-coated monodispersed magnetic poly(2-hydroxyethyl methacrylate) and poly(glycidyl methacrylate) microspheres by PCR

European Polymer Journal 68 (2015) 687–696

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Evaluation of poly(ethylene glycol)-coated monodispersed magnetic poly(2-hydroxyethyl methacrylate) and poly(glycidyl methacrylate) microspheres by PCR Daniel Horák a,⇑, Helena Hlídková a, Šteˇpánka Trachtová b, Miroslav Šlouf a, Bohuslav Rittich b, Alena Španová b a b

Institute of Macromolecular Chemistry, AS CR, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic Brno University of Technology, Faculty of Chemistry, Purkynˇova 118, 612 00 Brno, Czech Republic

a r t i c l e

i n f o

Article history: Received 24 September 2014 Received in revised form 18 February 2015 Accepted 16 March 2015 Available online 26 March 2015 Keywords: Magnetic microspheres Poly(ethylene glycol) Real-time PCR DNA isolation

a b s t r a c t New monodisperse magnetic poly(2-hydroxyethyl methacrylate) and poly(glycidyl methacrylate) microspheres were synthesized, coated with poly(ethylene glycol) and thoroughly characterized using several methods, such as scanning and transmission electron microscopy equipped with energy-dispersive X-ray spectroscopy (EDX), elemental analysis, ATR FT-IR and atomic absorption spectroscopy. The effect of different coatings of the microspheres on the DNA amplification was finally investigated using a real-time polymerase chain reaction (qPCR). The particles with a relatively high density of poly(ethylene glycol) and a low amount of carboxyl groups on the surface appeared suitable for DNA isolation. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Magnetic polymer microspheres are increasingly often used in many biomedical applications that are exemplified by cell manipulations [1,2], target ligand coupling [3], protein purification [4], nucleic acid capture [5], mRNA isolation [6,7], diagnostic assays [8] and therapeutic protocols [9]. The main advantage of these particles is their ability to respond to an external magnetic field, which enables one to work with large volumes and avoid time-consuming centrifugation or filtration. Thus, the magnetic separation is suitable for automated assays [10]. The separation is gentle, quick, simple and efficient and ensures reliable and reproducible results. Magnetic polymer microspheres were pioneered by Ugelstad, who was the first to made uniform polystyrene ⇑ Corresponding author. Tel.: +420 296 809 260. E-mail address: [email protected] (D. Horák). http://dx.doi.org/10.1016/j.eurpolymj.2015.03.036 0014-3057/Ó 2015 Elsevier Ltd. All rights reserved.

microspheres [11,12], which was previously only achieved using polymerizations under weightless conditions in space [13]. The only shortcoming was in the hydrophobic character of polystyrene particles, which were not amenable to further modification reactions, and the introduction of various required functional groups and ligands. In contrast, magnetic acrylamide [14], 2-hydroxyethyl methacrylate [15] or glycidyl methacrylate particles [16] have advantage of the increased hydrophilicity and easy modifiability. Therefore, the latter two monomers were chosen for the preparation of microspheres in this report. The major problem with biomedical applications of the magnetic polymer microspheres is connected with the undesirable nonspecific adsorption of proteins from cell lysates and complex aqueous media. The target molecule in the medium is usually available only at low concentrations and accompanied by high amounts of nonspecific compounds. Therefore, various blocking agents, e.g., albumin [17], zwitterionic phosphorylcholine [18] and

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non-charged polymers, such as poly(ethylene glycol) [19] and poly[N-(2-hydroxypropyl) methacrylamide] [20], were suggested to prevent the nonspecific adsorption. The polymerase chain reaction (PCR) is a well-known sensitive and rapid biochemical technique in molecular biology [21,22] and microbiology [23] in gene analysis for DNA cloning and, diagnosis of cancer [24,25], hereditary [26], infectious [27,28] and malignant diseases [29]. The PCR amplification of nucleic acids has become a powerful diagnostic tool to identify microorganisms in foods [30,31], clinical samples [32] and forensic analysis [33]. Because PCR amplifies the regions of the DNA that it targets, it can be used to analyze extremely small amounts of the samples [28]. In practice, PCR can fail from various reasons, such as its sensitivity to contaminations, which amplifies spurious DNA products. The presence of PCR inhibitors in DNA samples causes a number of problems, such as reduced amplification efficiency and assay sensitivity, or false-negative results. The negative effects of the inhibitors can be reduced by selecting the appropriate isolation method [34]. Numerous solid-phase systems, particularly based on magnetic particles, have been therefore suggested for DNA isolation [35–37]. Our previous results show that the magnetic core must be well covered by a robust polymer shell [38] to avoid interference of the particles with the PCR. Hence, PCR enables the verification of the incorporation of magnetic cores in the polymer nano- and microparticles [39]. The report was aimed to synthesize PEG-modified monodisperse magnetic poly(2-hydroxyethyl methacrylate) and poly(glycidyl methacrylate) microspheres and use PCR as a method to characterize the quality of microsphere coating.

2. Experimental 2.1. Materials 2-Hydroxyethyl methacrylate (HEMA; Röhm, Darmstadt, Germany), glycidyl methacrylate (GMA; Fluka; Buchs, Switzerland) and ethylene dimethacrylate (EDMA; Ugilor, France) were purified by vacuum distillation. 2-[(Methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA) and 2,20 -azobis(2,3,3-trimethylbuty ronitrile) were synthesized according to the published procedures [40,41]. Cyclohexyl acetate (CyAc) was prepared from cyclohexanol and acetic anhydride. Methocel 90 HG [(hydroxypropyl)methyl cellulose], dibutyl phthalate (DBP) and sodium dodecyl sulfate (SDS) were purchased from Fluka. FeCl2  4H2O, phosphate buffer (pH 7.4 and 8.1), bovine serum albumin (BSA) and bovine fibrinogen (Fg) were supplied by Sigma–Aldrich (St. Louis, USA), and bovine c-globulin (c-Gl) was supplied by Serva Electrophoresis (Heidelberg, Germany). Other chemicals were obtained from Lach-Ner (Neratovice, Czech Republic). All experiments were performed using Ultrapure Q-water, which was ultrafiltered in a Milli-Q Gradient A10 system (Millipore; Molsheim, France). DNA Escherichia coli (D4889; Sigma–Aldrich) was used as the DNA template in real-time PCR (qPCR). The PCR

primers were synthesized by Generi Biotech (Hradec Králové, Czech Republic); deoxyribonucleic acid sodium salt genomic, which was unsheared from E. coli strain B (D4889) and ethidium bromide were supplied by Sigma– Aldrich, and the DNA marker (100 bp ladder) for the gel electrophoresis was from Malamité (Moravské Prusy, Czech Republic). Agarose was purchased from Serva (Heidelberg, Germany). The commercially supplied ordinary chemicals and solvents were of analytical grade. 2.2. PEG-coated magnetic polymer microspheres Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA) particles were synthesized by multiple swelling polymerization of 2-hydroxyethyl methacrylate (40 wt.%), 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA; 20 wt.%) and ethylene dimethacrylate (EDMA; 40 wt.%) in the presence of inert solvents, such as CyAc and DBP [3]. Analogous poly(glycidyl methacrylate) (PGMA) microspheres were obtained by copolymerization of glycidyl methacrylate (50 wt.%), MCMEMA (10 wt.%) and EDMA (40 wt.%) which was followed by opening the reactive oxirane groups with ammonia to introduce the amino groups [17]. The MCMEMA moiety of both particles was hydrolyzed with NaOH to provide them with carboxyl groups. To render the particles with magnetic properties, the iron oxide was precipitated in the particle pores to produce magnetic PHEMA (mgtPHEMA) and magnetic PGMA microspheres (mgtPGMA) [42]. To reduce the undesirable nonspecific protein adsorption on the magnetic microspheres, they were modified with poly(ethylene glycol)s (PEG) of various molecular weights (Mw = 2000 and 5000) using EDC/NHS chemistry [43]. Although the mgtPHEMA particles were reacted with a-methoxy-x-aminoPEG5000 (Scheme 1a), their mgtPGMA counterparts were reacted with a,x-biscarboxyPEG2000 (Scheme 1b). Controlled amounts of carboxyl groups on the mgtPGMA microspheres were achieved using PEGylation with CH3O-PEG-NH2/H2N-PEG-NH-t-Boc mixture (100:1 and 1000:1 w/w; both Mw = 5000) under cloud point conditions [44–46]. During the reaction, the amino groups of the particles were transformed in carboxyls, the amino-terminated PEG was immobilized, excessive reactive groups were deactivated, tert-butoxycarbonyl (t-Boc) moieties were deprotected, and a controlled amount of carboxyl groups was introduced using succinylation [47]. The resulting microspheres were denoted as PEG5000mgtPHEMA, PEG2000-mgtPGMA (1.8 mmol COOH/g), PEG5000-mgtPGMA-1.8 (1.8 lmol COOH/g) and PEG5000mgtPGMA-18 (18 lmol COOH/g). 2.3. Characterization methods Scanning electron microscopy (SEM) was used to characterize the morphology and verify the composition of the microspheres. A Quanta 200 FEG SEM microscope (FEI; Brno, Czech Republic), which was equipped with an energy-dispersive X-ray (EDX) detector (EDAX; Mahwah, USA), was used to obtain secondary electron (SE) micrographs and EDX spectra. All micrographs and spectra were collected at an accelerating voltage of 30 kV. The size and

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Scheme 1. Surface modification of (a) mgtPHEMA and (b) mgtPGMA microspheres with PEG.

morphology of the microspheres were obtained from the SEM/SE micrographs. The size uniformity of the microspheres was characterized using the polydispersity index PDI = Dw/Dn, where Dw = RniD4/RniD3 and Dn = RniD/Rni; Dn and Dw are the number- and weight-average diameters, which obtained from the SEM micrographs by measuring at least 500 particles (Atlas software; Tescan; Brno, Czech Republic). The elemental composition of the magnetic microspheres was determined using EDX. The EDX spectra were collected from the microspheres that were deposited on a carbon support so that the iron oxide peaks did not overlap the peaks from the support. The elemental composition was calculated by the standardless ZAF method using the EDAX software. The EDX analysis from SEM, which is denoted as SEM/EDX, differentiated from the analogous TEM/EDX analyses that are described in the following paragraph. Transmission electron microscopy (TEM) was used to characterize the inner structure of the magnetic microspheres using bright field imaging (BF) to confirm the presence of iron oxide inside the microspheres (using EDX spectra) and to identify the iron oxide crystalline structure (using apertureless electron diffraction, ED). The samples for TEM were prepared by embedding the microspheres in epoxy LRW (London Resin White) resin and cutting them with a LKB III ultramicrotome (Leica Biosystems; Wetzlar, Germany) at room temperature. The ultrathin sections were collected on carbon-coated copper grids and investigated in a Tecnai G2 Spirit Twin 12 TEM microscope (FEI). All TEM micrographs, EDX spectra and ED diffractograms were collected at an accelerating voltage of 120 kV. The TEM/BF micrographs show a light-gray epoxy resin, medium-gray microspheres and dark-gray iron oxide nanoparticles. The EDX spectra (TEM/EDX) show the elemental composition of the individual microspheres, and the TEM/ED diffractograms show powder-like patterns, i.e., diffraction rings because of the presence of huge amounts of iron oxide nanocrystals in each micrograph. The TEM/EDX spectra were processed similarly to SEM/ EDX, but the composition was calculated using the thin sample-approximation (model M-thin). The two-dimensional TEM/ED diffractograms were processed and converted to one-dimensional diffraction patterns using the

ProcessDiffraction program [48] and compared with the theoretically calculated X-ray diffraction patterns (PowderCell program [49]) of known iron oxide structures, which are Fe3O4 and c-Fe2O3. The elemental analysis and Fe content in the bulk of the microspheres were determined using a Perkin–Elmer 2400 CHN elemental analyzer (Waltham, USA) and a Perkin– Elmer 3110 atomic absorption spectrometer (AAS). To compare the contents of carbon, hydrogen and nitrogen in the microspheres, the elemental analysis results were recalculated with assumption that Fe was absent from the particles. The FT-IR spectra were obtained on a Perkin–Elmer Paragon 1000PC spectrometer with a Specac MKII Golden Gate single attenuated total reflection (ATR) system. The microspheres were directly placed on the diamond crystal and measured with the angle of incidence of 45°. The wavenumbers ranged from 4400 to 450 cm1, the resolution was 4 cm1, and 64 scans were performed. Undesirable nonspecific adsorption on the microspheres was experimentally checked with bovine serum albumin (BSA), radiolabeled 125I-BSA, c-globulin, fibrinogen, pepsin and chymotrypsin as described in a previous paper [42]. 2.4. Real-time PCR Real-time PCR (qPCR) was performed using a qPCR2 SYTO-9 Master Mix (Top-Bio; Prague, Czech Republic). The DNA was amplified in a Rotorgene 6000 thermal cycler (Corbett Research; Mortlake, Australia) with Feub and Reub primers specific to the domain Bacteria [50]. The reaction involved 12.5 ll of qPCR 2 SYTO-9 Master Mix, 1 ll of each primer, 1 ll of the template DNA (100 ng ll1– 1 pg ll1), 4.5–9.5 ll of PCR-grade water and an appropriate volume of the particles. The final volume of the PCR mixture was adjusted to 25 ll. The amplification consisted of an initial cycle at 95 °C for 5 min, 30 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s and was completed with the fluorescence measurement. The amplification was followed by the melting-curve analysis of PCR products; the melting temperature (Tm) was 50–99 °C. A negative control (absence of DNA), a positive control (10 ng of DNA E. coli per ll) and a standard curve, which were

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Fig. 1. SEM micrographs of individual microspheres (a, b, d, e, g, h) and TEM micrographs of microsphere cross-sections (c, f, i) showing mgtPHEMA (a), PEG5000-mgtPHEMA (b, c), mgtPGMA (d), PEG2000-mgtPGMA (e, f), PEG5000-mgtPGMA-1.8 (g) and PEG5000-mgtPGMA-18 particles (h, i).

Table 1 Basic characteristics of magnetic PHEMA and PGMA microspheres.

a b c d

Microspheres

Dna (lm)

Cb (wt.%)

Hb (wt.%)

Nb (wt.%)

MgtPHEMA PEG5000-mgtPHEMA MgtPGMA PEG2000-mgtPGMA PEG5000-mgtPGMA-1.8c PEG5000-mgtPGMA-18d

3.9 4.0 5.0 5.0 5.3 5.3

46.5 46.8 45.6 47.4 47.5 47.7

5.9 6.6 6.2 6.5 6.2 6.4

0 0.31 1.48 1.57 1.52 1.62

Number-average diameter (SEM), PDI = 1.01. Elemental analysis. 1.8 lmol COOH/g. 18 lmol COOH/g.

generated by performing qPCR with a 10-fold serial dilution of E. coli DNA (100 ng ll1–1 pg ll1) were analyzed in each plate. The threshold cycle (Cq) was calculated as the cycle number where the fluorescence reached the threshold value (Tv) at the beginning of the exponential phase of the amplification curve. The amplification was calculated from the slope (M) of the calibration curve using the following equation:

r.e. = 10(1/M)  1, where r.e. is the reaction efficiency [51], which indicates the progress of the PCR reaction [52]. If the amplification efficiency is 1 (r.e. = 1), M = 3.33, which indicates that the amount of products doubles with each cycle [51]. The data were evaluated by the correlation coefficient R2 P 0.99 using the Rotorgene 6000 cycler software. The effect of the microspheres on the efficiency of amplification was determined at

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Germany). The mgnetic particles were separated on a DynaMag™-2 magnet (Life Technologies; Oslo, Norway).

3. Results and discussion 3.1. PEGylated magnetic polymer microspheres: Morphology, size and composition

Fig. 2. Typical SEM/EDX spectrum of PEG5000-mgtPGMA-18 microspheres (18 lmol COOH/g).

several particle concentrations. The amount of PEG5000mgtPHEMA, PEG2000-mgtPGMA, PEG5000-mgtPGMA-1.8 and PEG5000-mgtPGMA-18 particles in the PCR mixture (25 ll) was 0.05–50 lg. Agarose gel electrophoresis was performed on a Bio-Rad device (Richmond, USA), and the PCR products were visualized on a UltraLum EB-20E UV transilluminator (Paramount, USA) at 305 nm and photographed with a digital camera. The UV spectrophotometry of DNA was performed on a UV/VIS NanoPhotometer (Implen; München,

To investigate the DNA isolation from the model solutions, newly developed PEG-mgtPHEMA and PEGmgtPGMA microspheres [44] were selected as a probe in qPCR. The initial monodisperse magnetic PHEMA particles were 3.9 lm in diameter and contained 1 mmol COOH/g. Similarly, the monodisperse magnetic PGMA microspheres were 5 lm in size and contained 0.6 mmol COOH and 1.2 mmol NH2 per g [47]. The size and morphology of the neat magnetic, PEGmgtPHEMA and PEG-mgtPGMA microspheres were visualized in SEM/SE micrographs (Fig. 1a, b, d, e, g, h). The SEM of the PEG5000-mgtPHEMA particles (Fig. 1b) does not show any noticeable change compared with the neat mgtPHEMA ones (Fig. 1 a). Similarly, only a slightly smoother surface accompanied with small aggregates of PEG chains is visible on the PEG-coated magnetic PGMA particle surface (Fig. 1e, g, h) compared with the initial mgtPGMA (Fig. 1d). The particle surface was analyzed in more details using more advanced techniques, such as

Fig. 3. Experimental electron diffraction pattern (TEM/ED) of PEG5000-mgtPGMA-18 microspheres (a) and its comparison with calculated XRD diffraction patterns of c-Fe2O3 and Fe3O4 (b). PEG5000-mgtPGMA-18 microspheres are quickly attracted by a magnet (c).

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Fig. 4. ATR FT-IR spectra of neat PHEMA (a), mgtPHEMA (b), PEG5000mgtPHEMA microspheres (c) and neat CH3O-PEG5000-NH2 (e). Differential spectrum of PEG5000-mgtPHEMA minus mgtPHEMA particles (d).

AFM, which displays dense surface PEG coverage on both PEG5000-mgtPGMA-1.8 and PEG5000-mgtPGMA-18 microspheres [42]. The number-average diameters Dn of PEGylated mgtPHEMA and mgtPGMA microspheres slightly increase (Table 1). All PEG-modified magnetic microspheres remain monodisperse with PDI = 1.01 (Table 1). The presence of iron oxide nanoparticles inside the PEGylated microspheres was documented using TEM/BF micrographs of the cross-sections (Fig. 1c, f, i) and TEM/

EDX and SEM/EDX spectra (Fig. 2). The TEM/BF micrographs show that the dispersion of iron oxide nanoparticles in the polymer microspheres is notably homogeneous, even if the nanoparticles form small agglomerates. The TEM/ED diffractograms (Fig. 3a) and their analysis (Fig. 3b) show that all PEGylated magnetic microspheres have identical diffraction patterns, which correspond to the magnetic form of iron oxide (either cFe2O3 (maghemite) or Fe3O4 (magnetite)). The crystalline structures and consequently the diffraction data of cFe2O3 or Fe3O4 are notably similar, and the experimental TEM/ED patterns cannot reliably differentiate these two forms. A slightly better consistency between the calculated XRD pattern and the experimental ED data was obtained for c-Fe2O3 (Fig. 3b). In any case, TEM/ED definitely excludes the presence of the non-magnetic forms of iron oxide. The presence of magnetic iron oxide nanoparticles inside the polymer microspheres, which was unambiguously proved using microscopic methods, results in sufficient magnetic properties of the particles that can be easily separated in a magnetic field [17] as documented in Fig. 3c. A quantitative analysis of the Fe amount in the magnetic microspheres, which was 14–18 wt.%, was obtained from AAS. The presence of the PEG coating on the microspheres was documented using an elemental analysis because both nitrogen and carbon contents in PEG-mgtPHEMA and PEGmgtPGMA increased compared with those of nonPEGylated particles (Table 1). It should be noted that both elemental analysis and AAS provided more precise data than the SEM(TEM)/EDX spectra, which show notably high carbon peaks because of the signal from the graphite support and/or carbon film. Moreover, the EDX analysis lacked the standards. Thus, it can be concluded that the EDX

Fig. 5. Dependence of DNA amplification on the amount of the microspheres in the PCR mixture. The 10-fold serial dilutions of Escherichia coli DNA (100 ng ll1-1 pg ll1) were analyzed in each plate in the presence of PEG5000-mgtPGMA-1.8 particles. (a) 0, 0.85, 1.7, 4.25 and 8.5 lg particles/25 ll PCR mixture; (b) 0, 0.09, 0.17, 0.43 and 0.85 lg particles/25 ll PCR mixture.

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D. Horák et al. / European Polymer Journal 68 (2015) 687–696 Table 2 Effect of the magnetic microspheres on inhibition of DNA amplification in qPCR. Magnetic microspheres

Weight of particlesa (lg)

r.e.b (%)

R2c

Fluorescence inhibitiond

Specific PCR product detectiond

PEG5000-mgtPHEMA

0 0.05 0.10 0.25 0.50 1 2.5 5 10 25 50

96 84 85 84 89 76 49 n.d. n.d. n.d. n.d.

0.99 0.99 0.99 0.99 0.90 0.98 0.73 n.d. n.d. n.d. n.d.

     + + + + + +

+ + + +       

PEG2000-mgtPGMA

0 0.05 0.10 0.25 0.50 1 2.5 5

87 85 84 84 89 n.d. n.d. n.d.

0.99 0.99 0.99 0.99 0.69 n.d n.d. n.d.

     + + +

+ + + + +   

PEG5000-mgtPGMA-1.8e

0 0.08 0.17 0.43 0.85 1.7 4.25 8.5

87 88 88 90 90 92 242 n.d.

0.99 0.99 0.99 0.99 0.99 0.98 0.30 n.d.

      + +

+ + + + + + + 

PEG5000-mgtPGMA-18f

0 0.07 0.13 0.33 0.65 1.3 3.25 6.5

88 90 85 84 94 93 101 n.d.

0.99 0.99 0.99 0.99 0.98 0.99 0.87 n.d.

       +

+ + + + + + + +

n.d. not detected. a In 25 ll of the PCR mixture. b Reaction efficiency. c Correlation coefficient. d ‘‘+’’ inhibition/specific PCR products detection, ‘‘’’ no inhibition effect/no specific PCR product detection. e Particles contained 1.8 lmol COOH/g. f Particles contained 18 lmol COOH/g.

spectra can only be used for the local semi-quantitative determination of the particle composition and confirmation of the AAS results. The binding of PEG5000 chains to the mgtPHEMA microspheres (analogously to the mgtPGMA; data not shown) was studied using ATR FT-IR spectroscopy (Fig. 4). The neat (non-magnetic) and mgtPHEMA microspheres have similar spectra. In the latter spectrum, a new broad asymmetric band at ca. 560 cm1 appears because of Fe–O stretching vibrations [53] which confirms the formation of c-Fe2O3 inside the microspheres [54,55]. Moreover, there is a strong asymmetrical stretching band of carboxyl anion at 1585 cm1, which originates from the asymmetrical NH+3 bending accompanied with a weak symmetrical 1533 cm1 band of NH+3 bending. This result confirms that ammonia reacts with the COOH groups of the particles and forms primary amino groups during the c-Fe2O3

precipitation [56,57]. After the particle modification with PEG, only slight changes were noticeable in the FT-IR spectrum (Fig. 4). They include two peaks (strong and weak at 1603 and 1322 cm1, respectively), which are assigned to the amide II band of the primary or secondary amides (N–H bending) and amide III band (C–N stretching), respectively. Thus, formation of the CO–NH bonds and the modification of the magnetic PHEMA particles with PEG were confirmed. Because of possible shielding effects, the differential spectrum (PEG-modified minus unmodified magnetic particles) was included in Fig. 4. A few more peaks at 1541, 1263 (amide II and III bands of the transsubstituted secondary amide with interaction between the N–H bending and C–N stretching vibration of C–N–H) and 705 cm1 (out-of-plane N–H wagging) were observed in the differential spectrum. The FT-IR spectra of PGMA particles are shown elsewhere [47].

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3.2. Effect of magnetic particles on the DNA amplification in qPCR The qPCR based on the quantification of Cq is notably sensitive to the presence of any inhibitors in the reaction mixtures [58]. The deviation of the amplification efficiency from 1 provided a measure of the PCR inhibition with r.e. 0.8–1.1 (M between 3.9 and 3.0), which is acceptable [39,59]. Generally, increasing the inhibitor concentration decreases the DNA amplification efficiency and reaction curve slope [39,60]. The amplification plot of the number of cycles versus the fluorescence signal correlated with the initial amount of target DNA (100 ng ll1–1 pg ll1) in the presence of PEG5000-mgtPGMA-1.8 particles in concentration 0–8.50 lg/25 ll PCR mixture (Fig. 5) (see Table 2). The effect of the magnetic PHEMA and PGMA microspheres on the inhibition of DNA amplification in qPCR is shown in [39]. Increasing the concentration of magnetic particles in the PCR mixture increases the inhibition effect. It should be noted that the particles affect the PCR source by extinguishing the fluorescence of fluorescent dye (Fig. 5), but the PCR product was synthesized as confirmed by the conventional PCR with the gel electrophoresis. Strong inhibition was observed with the PEG5000mgtPHEMA microspheres most likely because of their incomplete coating with PEG using the EDC/NHS chemistry. In addition, the PEG2000-mgtPGMA particles did not substantially increase the amplification effect. In contrast, the PEG5000-mgtPGMA-1.8 and PEG5000-mgtPGMA-18

particles, which were coated under cloud point conditions, only mildly inhibited the DNA amplification because of the presence of dense PEG coating on the particle surface. Thus, a reduced concentration of COOH groups is beneficial to DNA amplification. The amplicon specificity was determined based on the melting-curve analysis of the qPCR products (466 bp) at 89 °C in the presence of various amounts of PEG5000mgtPGMA-1.8 particles (Fig. 6). The qPCR results are consistent with the detection of specific PCR products of domain Bacteria (466 bp) using the agarose gel electrophoresis [50,61] (Fig. 7). 4. Conclusions New monodisperse magnetic PHEMA and PGMA microspheres were developed by modifying Ugelstad’s process. The presence of iron oxide inside the microspheres was proved using TEM/BF micrographs, SEM and TEM/EDX spectra, TEM/ED diffractograms and atomic absorption spectroscopy. Successful coating of the magnetic microspheres with PEG (inert noninterfering polymer) was confirmed with elemental analysis and FT-IR spectroscopy. qPCR based on the shift of Cq values after adding different amounts of the particles to the DNA was found as a suitable method to evaluate their inhibition effect. If the magnetic particles are poorly coated with the PEG, qPCR is negatively affected. It can be assumed that a denser PEG coating corresponds with lower DNA inhibition in qPCR.

Fig. 6. Melting curve analysis of qPCR products (466 bp) at 89 °C after amplification of DNA in the presence of various amounts of PEG5000-mgtPGMA-1.8 microspheres. The 10-fold serial dilutions of Escherichia coli DNA (100 ng ll1–1 pg ll1) were analyzed in each plate. (a) 0, 0.85, 1.7, 4.25 and 8.5 lg particles/25 ll PCR mixture; (b) 0, 0.09, 0.17, 0.43 and 0.85 lg particles/25 ll PCR mixture.

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Fig. 7. Gel electrophoresis of qPCR products specific to domain Bacteria (466 bp) in the presence of PEG5000-mgtPGMA-1.8 in 25 ll of the PCR mixture. The 10-fold serial dilution of Escherichia coli DNA (100 ng ll1–1 pg ll1) were analyzed in each plate. (a) 1–6: 0 lg microspheres, 7: marker 100 bp, 8–13: 0.08, 15–20: 0.17, 22–27: 0.42 and 29–34: 0.85 lg microspheres, 35: marker 100 bp and 36: negative control. (b) 1–6: 0 lg microspheres, 7: marker 100 bp, 8– 13: 0.8, 15–20: 1.7, 22–27: 4.2 and 29–34: 8.5 lg microspheres, 35: marker 100 bp and 36: negative control.

Another factor that affects the qPCR is the amount of carboxyl groups on the particle surface. More carboxyl groups on the particles correspond to higher PCR inhibition. This result is consistent with previous reports, where nonspecific protein sorption is directly proportional to the concentration of carboxyl groups in the support [42,47]. The PEG5000-mgtPGMA-18 microspheres that are coated under the cloud point conditions have high reaction efficiency and only slightly inhibit the DNA amplification. Thus, such particles can be recommended for the immunomagnetic isolation of DNA, which is important for the early detection of many inherited and malignant diseases. Notes The authors declare no competing financial interest. Acknowledgements Financial support of the Grant Agency of the Czech Republic (grant P206/12/0381 and 13-3284 OP) and Ministry of Education, Youth and Sports of the Czech Republic (project LH14318) is gratefully acknowledged. References [1] Kuan WC, Horák D, Plichta Z, Lee WC. Immunocapture of CD133positive cells from human cancer cell lines by using monodisperse magnetic poly(glycidyl methacrylate) microspheres containing amino groups. Mater Sci Eng C 2014;34:193–200. [2] Wadajkar AS, Santimano S, Tang L, Nguyen KT. Magnetic-based multi-layer microparticles for endothelial progenitor cell isolation, enrichment, and detachment. Biomaterials 2014;35:654–63.

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