chemosensors

Article Immunomagnetic Separation of Microorganisms with Iron Oxide Nanoparticles

Julian A. Thomas, Florian Schnell, Yasmin Kaveh-Baghbaderani , Sonja Berensmeier and Sebastian P. Schwaminger * Bioseparation Engineering Group, Department of Mechanical Engineering, Technical University of Munich, 85748 Garching, Germany; [email protected] (J.A.T.); [email protected] (F.S.); [email protected] (Y.K.-B.); [email protected] (S.B.) * Correspondence: [email protected]; Tel.: +49-89289-15747



Received: 7 February 2020; Accepted: 24 February 2020; Published: 27 February 2020


Abstract: The early detection of Legionella in water reservoirs, and the prevention of their often fatal diseases, requires the development of rapid and reliable detection processes. A method for the magnetic separation (MS) of Legionella pneumophila by superparamagnetic iron oxide nanoparticles is developed, which represents the basis for future bacteria detection kits. The focus lies on the separation process and the simplicity of using magnetic nanomaterials. Iron oxide nanoparticles are functionalized with epoxy groups and Legionella-specific antibodies are immobilized. The resulting complexes are characterized with infrared spectroscopy and tested for the specific separation and enrichment of the selected microorganisms. The cell-particle complexes can be isolated in a magnetic field and detected with conventional methods such as fluorescence detection. A nonspecific enrichment of bacteria is also possible by using bare iron oxide nanoparticles (BIONs), which we used as a reference to the nanoparticles with immobilized antibodies. Furthermore, the immunomagnetic separation can be applied for the detection of multiple other microorganisms and thus might pave the way for simpler bacterial diagnosis.

Keywords: magnetic nanoparticles; iron oxide nanoparticles; immunoassay; magnetic separation; microorganism detection; nanoparticle functionalization; antibody immobilization

1. Introduction Fast bacterial separation methods are crucial for diagnostics, and the development of rapid detection and quantification devices. Novel methodologies are needed and can reduce the diagnostic time and thus allow faster treatment. Legionella pneumophila is a pathogen, which causes Legionnaire’s disease, which are quite difficult to cultivate, which leads to detection times for contaminated water sources of up to 14 days. This timeframe should be reduced since Legionnaire’s disease is an “avoidable” disease, which is not passed on from person to person but which is fatal in 5–40% of cases [1,2]. In addition to detection in water, the rapid and accurate detection of Legionella in body fluids is essential for early medical diagnosis. To date, 50 Legionella species and more than 70 serogroups are known. More than 90% of the cases of legionnaire’s disease are caused by Legionella pneumophila which is a rod-shaped bacterium with 0.4–1.5 µm diameter and up to 6 mm length [1,3]. The standardized cultivation of Legionella is regulated internationally by ISO 11371. The typical determination of a Legionella contamination is based on the sampling of 100–1000 mL water and the cultivation of the microorganisms on a GVPC (Glycine Vancomycin Polymyxin Cycloheximide) agar, which takes 10 to 14 days due to the long doubling time of Legionella pneumophila in order to determine the limit value of 100 colony forming unit in 100 mL of water. While this quite time-consuming technique still represents the state of the art, faster processes have been reported but are often either quite complicated

Chemosensors 2020, 8, 17; doi:10.3390/chemosensors8010017 www.mdpi.com/journal/chemosensors Chemosensors 2020, 8, 17 2 of 12 or expensive [4–6]. These methods include fluorescent antibody staining, antigen detection, serological testing and polymerase chain reaction (PCR) [5,7]. Filtration, centrifugation, and immunomagnetic separation (IMS) techniques can be used to concentrate bacteria and are often necessary in order to achieve a higher detectability of the pathogens and to condition the samples [8–10]. Pre-purification steps are essential in the separation of Legionella from greywater [11]. The separation of a sample mixture by IMS can significantly accelerate the detection process by concentrating the target bacterium and removing interfering substances [12]. For this, neither expensive sophisticated equipment nor scientific knowhow is required. IMS is based on the separation of adsorbent from the liquid phase in a magnetic field. Thus, superparamagnetic iron oxide nanomaterials with a high specific surface area are a promising adsorbent material. Bare iron oxide nanoparticles (BIONs) with 5–50 nm size can be used for e.g. protein purification [13,14], enzyme immobilization [15] and harvesting of algae [16]. Usually, the nanoparticles are embedded in polymers and used as superparamagnetic microbeads (0.1–100 µm) and are further modified with functional group to anchor an immunoactive group, such as an antibody [17]. The immobilized antibody needs to be specific to the respective epitope on the surface of Legionella pneumophila. Subsequent fluorescence analysis e.g. with a fluorescent-labelled antibody offers the potential for rapid and accurate quantification [18]. In the long term, a kit could be developed that enables the detection of Legionella by untrained staff on site and in a few hours. Similar approaches either use magnetic beads which recognize antibodies coupled to the Legionella pneumophila [19,20], or, a cell-specific antibody coupled to the magnetic beads [18,21]. Bloemen et al. used 5–25 µm sized PEG-coated magnetic beads with immobilized antibodies for the separation of Legionella and needed a contact time of 1 h [21]. The particle size significantly influences the separation efficiency. Of crucial importance in the evaluation of a separation process is the recovery rate, which relates to amount of cells which are successfully separated from the original solution. Using commercial MACS®microbeads coated with monoclonal mouse anti-FITC antibodies, Füchslin et al. reached a separation efficiency of 95% of the Legionella in an aqueous suspension. Then, 92% Legionella cells could be separated from a cell mixture with Escherichia coli, while only adsorbing below 1% of E. coli cells [19]. Other approaches to separate Legionella include self-synthesized Fe3O4-gold-particles and commercial streptavidin-coated magnetic nanoparticles [22]. The goal of this work is the successful functionalization of magnetic nanoparticles with antibodies for the separation of the bacteria in an aqueous medium. The magnetic nanoparticles used for the immobilization of antibodies are synthesized and characterized for the present work. For this purpose, the size of particles were determined with dynamic light scattering (DLS), the zeta potential was investigated as a function of the pH-value, and an analysis of the crystal structure was carried out with X-ray diffraction (XRD). The success of the coating and coupling processes is verified by IR spectroscopy. Furthermore, the magnetization of the particles is investigated. Magnetic separation can be used as a preliminary step for subsequent filtration or direct detection using further labelling or cultivation. The elaborated separation method was tested for its transferability to the separation of other cell types and its specificity to Legionella. Therefore, we compared the nanoparticles with bare iron oxide nanoparticles (BIONs). Since even those non-functionalized nanoparticles show a high binding affinity, we further tested the influence of pH on the enrichment of bacteria. The aim is a quick and easy separation and enrichment process, which nevertheless provides reliable qualitative results.

2. Materials and Methods

2.1. Synthesis In accordance with Roth et al., 35.0 g of iron (II) chloride and 86.4 g of iron (III) chloride, obtained from Sigma-Aldrich, were dissolved and mixed in 200 mL of deionized water [23]. 1 M NaOH (Fluka) was prepared with deionized water and filled in a stirred tank glass reactor under a nitrogen atmosphere [23]. Subsequently, the salt solution was added dropwise, which immediately forms a black precipitate. After complete addition, the reaction was continued for half an hour under constant Chemosensors 2020, 8, 17 3 of 12 conditions. The resulting particles were washed several times with deionized water until a conductivity of lessChemosensors than 200 2020µ,S 7/,cm x FOR was PEER reached. REVIEW 3 of 12

2.2.blac Coatingk precipitate. After complete addition, the reaction was continued for half an hour under constant conditions. The resulting particles were washed several times with deionized water until a conductivityBIONs (1 g) of areless dispersedthan 200 μS/cm in 300 was mL reached. of ethanol in a round bottom flask and sonicated for 1 h. After the addition of 3-aminopropyltriethoxysilane (APTES) followed the mixture was treated another hour2.2. in Coating an ultrasonic bath. The reaction product was dispersed four times in ethanol for washing and then separatedBIONs (1 magnetically. g) are dispersed It was in 300 then mL washed of ethanol inwater in a round until bottom a conductivity flask and of sonicated less than for 200 1 h.µ S/cm wasAfter measured. the addition Subsequently, of 3-aminopropyltriethoxysilane 25 mg of the functionalized (APTES) particles followed were the mixed mixture with was 2 mL treated of 2% (v/v) glutaraldehyde.another hour Ain pHan valueultrasonic of 11 bath. was The set byreaction titration product of 0.5 was M NaOH. dispersed During four thetimes titration, in ethanol the solutionfor is stirredwashing constantly and then for separated 1 h. The magnetically. resulting polyglutaraldehyde It was then washed (PGA)-coated in water until particlesa conductivity were washedof less ten timesthan in 200 water, μS/cm twice was in measured. 1 M NaCl Subsequently, solution, and 25 mg another of the twofunctionalized times in water. particles The were complete mixed with washing process2 mL was of 2 repeated% (v/v) glutaraldehyde. twice. A pH value of 11 was set by titration of 0.5 M NaOH. During the titration,The washed the solution particles is stirred were thenconstantly suspended for 1 h.at The a concentrationresulting polyglutaraldehyde of 25 g/L in a (PGA) solution-coated of 0.5 M particles were washed ten times in water, twice in 1 M NaCl solution, and another two times in water. NaOH, 19 mM NaBH4, and 5% (v/v) epichlorhydrin. The mixture reacts under continuous stirring The complete washing process was repeated twice. for 6 h. During the reaction, epichlorhydrin binds to the oxygen of the aldehyde and epoxy groups The washed particles were then suspended at a concentration of 25 g/L in a solution of 0.5 M are formed. Afterwards, the product was washed eight times with deionized water and twice with a NaOH, 19 mM NaBH4, and 5% (v/v) epichlorhydrin. The mixture reacts under continuous stirring buffforer solution6 h. During (20 the mM reaction, NaH2PO epichlorhydrin4, 1.0 M NaCl, bind pHs =to6.8). the oxygen Until further of the aldehyde use, theparticles and epoxy were groups stored at 4 ◦Care in formed. the same Afterwards, buffer solution. the product The was PGA-coated washed eight particles times with deionized functional water epoxy and groups twice with are a called @Epoxybuffer in solution the further (20 mM text. NaH2PO4, 1.0 M NaCl, pH = 6.8). Until further use, the particles were stored at 4 °C in the same buffer solution. The PGA-coated particles with functional epoxy groups are called 2.3.@Epoxy Particle in Antibody the further Coupling text. Specific rabbit anti-Legionella antibodies (PA1-7227) from Thermo Fisher Scientific are immobilized 2.3. Particle Antibody Coupling on @Epoxy particles. The coupling is achieved by direct contact between nanoparticles and proteins. The antibodiesSpecific are rabbit coupled anti-Legionella through antibodies the epoxy (PA1group-7227) tothe from @Epoxy Thermo particles. Fisher ScientificTo determine are the amountimmobilized of antibody on @Epoxy that can particles be bound. The by coupling the coated is achie nanoparticles,ved by direct ancontact adsorption between test nanoparticles was performed and proteins. The antibodies are coupled through the epoxy group to the @Epoxy particles. To with bovine serum albumin and antibodies, which yielded optimal binding conditions at 0.2 mg/mL determine the amount of antibody that can be bound by the coated nanoparticles, an adsorption test for a nanoparticle concentration of 1 mg/mL. Thus, for the bacterial enrichment experiments, 1 mL was performed with bovine serum albumin and antibodies, which yielded optimal binding of antibodyconditions suspension at 0.2 mg/mL (c = for0.2 a mg nanop/mL)article was addedconcentration to 1 mg of nanoparticles. 1 mg/mL. Thus, After for the incubation bacterial with heat-inactivatedenrichment experiments,Legionella,obtained 1 mL of from antibody BacTrace suspension GmbH, (c the = particle 0.2 mg/mL fraction) was isadded centrifuged to 1 mg off and thenanoparticles residual concentration. After incubation of Ab with in the heat supernatant-inactivated Legionella is measured, obtained by two from methods. BacTraceA GmbH bicinchoninic, the acidparticle (BCA) fraction assay was is centrifuged carried out off toand determine the residual the con proteincentration concentration. of Ab in the supernatant In addition, is measured samples were analyzedby two by methods. UV/vis spectroscopyA bicinchoninic at 230 acid nm (BCA) and 280assay nm. was carried out to determine the protein concentration.The whole synthesis, In addition, coating, samples functionalization, were analyzed by and UV/vis antibody spectroscopy coupling at 2 procedure30 nm and is280 illustrated nm. in Scheme1The. whole synthesis, coating, functionalization, and antibody coupling procedure is illustrated in Scheme 1.

+ Antibodies

+ APTES + PGA and Epichlohydrin

SchemeScheme 1. Schematic 1. Schematic illustration illustration of theof the iron iron oxide oxide synthesis, synthesis, APTES APTES coating, coating, polyglutaraldehyde polyglutaraldehyde (PGA) functionalization,(PGA) functionalization, epichlorhydrin epichlorhydrin activation, activation and antibody, and antibody coupling. coupling.

Chemosensors 2020, 8, 17 4 of 12

2.4. Characterization Dynamic light scattering (DLS) and zeta potential measurements were performed with a Beckman Coulter Delsa Nano C Particle Analyzer. The pH is varied by titration of 0.1 M HCl and 0.1 M NaOH. The characterizations are typically performed at a concentration of 1 g/L of nanoparticles in deionized water. The Fourier-transform infrared spectra (FTIR) were measured using a Bruker ALPHA II FTIR spectrometer and the matching Platinum attenuated total reflection (ATR) module. Sixty-four scans per sample and measurement were performed and a baseline was subtracted via the rubber band method in the software OPUS. Transmission electron microscopy (TEM) was carried out with the JEM 1400 Plus microscope from JEOL and the recorded images were subsequently evaluated using ImageJ software. Diluted nanoparticle suspensions were precipitated on carbon copper grids prior to TEM measurements. Around 100 particles per nanoparticle type were measured. X-ray diffraction (XRD) was recorded with a STOE Stadi P diffractometer with a molybdenum radiation source from STOE and Cie. The freeze-dried samples were mounted in transmission geometry and measured while rotating. The magnetic susceptibility of the nanoparticles is determined by a SQUID characterization. For this work, the Quantum Design MPMS 5XL SQUID magnetometer was used. The measurements were carried out at 300 K in magnetic fields varying between 50 kOe to 50 kOe. − 2.5. Binding of Cells to the Nanoparticles Next, 0.8 mg/mL magnetic nanoparticles were added to the cell suspension in phosphate buffered saline (PBS) buffer (20 mM NaH2PO4, 1.0 M NaCl, pH = 6.8) and incubated in a shaker for at least 1 h. Depending on the nature of the particles and the use of antibodies, different amounts of cells were bound and magnetically separated. In the case of antibody-particle complexes, 0.1 gAb/gParticles were used. The number of bound cells were determined by measuring the absorbance at 600 nm in the supernatant. To estimate the number of cells that can be separated by BIONs, a series of experiments with different nanoparticle concentrations were performed. The nanoparticles are added to 1 mL samples of suspensions of E. coli BL21(DE3) with an optical density of 0.2. This corresponds to 5 107 × cells [24]. The mass of BIONs varied from 0.4 mg to 16 mg. In order to investigate the specificity of the antibodies, 0.8 mg @Epoxy+Ab complexes were added to each 1 mL of different cell suspensions (E. coli, Legionella pneumophila, OD600 = 0.2). After 1 h coupling, the particle fraction was separated magnetically. To examine the influence of the pH on the magnetic separation, in each case 1 mL of the PBS buffered (20 mM) E. coli was centrifuged off and resuspended in a series of buffers of different pH values. Subsequently, 16 mg of BIONs were added, which are dissolved in 1 mL of the same buffer. The mixture is incubated for 1 h in a shaker at room temperature. Subsequently, the cell-particle complexes are magnetically separated over 1 h. The absorbance of the supernatant is measured. In addition, the OD600 of analogously prepared suspensions without nanoparticles were measured. Incubation experiments were performed in triplicates.

3. Results and Discussion Nanoparticles are characterized towards their size and material properties in order to facilitate a proper immunomagnetic separation. Figure1 shows images of the particles taken via TEM. The frequency distributions are in good agreement with previous measurements of magnetite particles [15,21,25–27]. Here, the actual dimensions of the individual particles can be determined optically, whereas dynamic light scattering yields the hydrodynamic diameter of agglomerates [17]. The BIONs show an approximately Gaussian size distribution with a maximum around 10 nm, while the coated and polyglutaraldehyde activated nanoparticles show a broader size distribution with a higher maximum around 12 nm. The difference between the TEM diameters can be related to the thickness of the coating. Chemosensors 2020, 7, x FOR PEER REVIEW 5 of 12

50 BIONs @Epoxy 40

30

20 Particle count Particle 10

0 5 7 9 11 13 15 17 19 21 Particle diameter (nm) (a) (b) (c)

Figure 1. Transmission electron microscopy (TEM)-microphotography of bare iron oxide nanoparticles (BIONs) (a) and of @Epoxy particles (b). Frequency distribution of pure BIONs and PGA-coated SPIONs with various particle diameters, measured by TEM (c).

Aside from the size of nanoparticles, hydrodynamic properties play an important role for the Chemosensorsfurther coating2020, 8 ,process 17 and the functionalization of iron oxide nanoparticles with antibodies. Figure5 of 12 Chemosensors 2020, 7, x FOR PEER REVIEW 5 of 12 2a shows a dynamic measurement of the zeta potential of pure uncoated nanoparticles. The potential reaches values from +17.04 mV at pH = 3.1 up to −34.42 mV at pH = 10.91. The isoelectric point of BIONs is at about pH = 6, which matches the results of Bini et al. and Schwaminger et al. [27,28]. 50 The charge of the particles can be explained by hydroxyl groups on the surface of the BIONs BIONs. @Epoxy These can be detected by FTIR spectroscopy. In the basic range, the Fe-OH40 group dissociates to form

Fe-O . In the acidic range, a proton is taken up and Fe-OH is formed30 [29]. The stabilization of nanoparticles at alkaline pH values is necessary in order to obtain a homogeneous coating and thus 20

a further functionalization with APTES. Stable non-agglomerated BIONs form the basis of the further Particle count Particle functionalization and antibody immobilization. APTES coated nanoparticles10 show a zeta potential of 17.59 at pH 6.4 and the @EpoxyPGA functionalized nanoparticles possess0 a zeta potential of −23.88 5 7 9 11 13 15 17 19 21 at pH 7.3, which further indicates a successful coating and functionalization. Particle diameter (nm) Figure 2b (showsa) the DLS-measured size ( distributionb) of the BIONs at pH(c ) 10, where the nanoparticles are colloidally stable with aggregated structures of ~80 nm size and possess a high zeta potentialFigureFigure (>30 1. Transmission 1. mV) Transmission [25]. electron electron microscopy microscopy (TEM)-microphotography (TEM)-microphotography of bare iron of oxide bare nanoparticles iron oxide (BIONs)Thenanoparticles silica (a) and coating (BIONs of @Epoxy ) and(a) and particles the of polyglutaraldehyde@Epoxy (b). Frequency particles (b distribution). Frequency activation of distribution purestabilize BIONs of the and pure particles PGA-coated BIONs and against agglomeration,SPIONsPGA-coated with whi variousSPIONsch explains particle with various diameters, the smallerparticle measured diameters,hydrodynamic byTEM measured (c ).diameters by TEM (~55(c). nm). The stabilization is causAsideed by the from mutual the size repulsion of nanoparticles, of electrostatically hydrodynamic charged properties particles [28] play. an important role for the Aside from the size of nanoparticles, hydrodynamic properties play an important role for the furtherNevertheless, coating process the and values the functionalization measured for the of iron coated oxide particles nanoparticles overestimate with antibodies. the actual Figure particle2a further coating process and the functionalization of iron oxide nanoparticles with antibodies. Figure showsdiameter. a dynamic This is measurement due to still-existing of the zeta agglomeration potential of pureeffects, uncoated and liquid nanoparticles. boundary The layers, potential which 2a shows a dynamic measurement of the zeta potential of pure uncoated nanoparticles. The potential reachessurround values the particles from +17.04 in the mV aqueous at pH =phase.3.1 up A tomore34.42 accurate mV at determination pH = 10.91. The of the isoelectric average pointsize of of a reaches values from +17.04 mV at pH = 3.1 up to− −34.42 mV at pH = 10.91. The isoelectric point of BIONssingle isparticle at about is possible pH = 6, whichby TEM matches [26]. the results of Bini et al. and Schwaminger et al. [27,28]. BIONs is at about pH = 6, which matches the results of Bini et al. and Schwaminger et al. [27,28]. The charge of the particles can be explained by hydroxyl groups on the surface of the BIONs. 40 These can be detected by FTIR spectroscopy. In the basic range,16 the Fe-OH group dissociates BIONs to form 30 @Epoxy Fe-O . In the acidic range, a proton is taken up and Fe-OH14 is formed [29]. The stabilization of 20 nanoparticles at alkaline pH values is necessary in order to 12obtain a homogeneous coating and thus 10 10 a further functionalization0 with APTES. Stable non-agglomerated BIONs form the basis of the further 8 functionalization and antibody immobilization. APTES coated nanoparticles show a zeta potential of -10 6 17.59 at pH-20 6.4 and the @EpoxyPGA functionalized nanoparticles possess a zeta potential of −23.88

Zetapotential (mV) 4 Numberdistribution (%) at pH 7.3-30, which further indicates a successful coating and functionalization.2 Figure-40 2b shows the DLS-measured size distribution0 of the BIONs at pH 10, where the 2 4 6 8 10 12 10 100 1000 nanoparticles are colloidallypH stable with aggregated structures of ~80Hydrodynamic nm size diameter and possess (nm) a high zeta potential (>30 mV) [25]. The silica coating( a) and the polyglutaraldehyde activation stabilize(b) the particles against agglomeration, which explains the smaller hydrodynamic diameters (~55 nm). The stabilization is causFigureed by 2.the(a )mutual Zeta potential repulsion of BIONs of electrostatically at various pH-values charged titrated particles from [28] pH. 3 to pH 11 with sodium

hydroxideNevertheless, (0.1 M). the (b) Size values distribution measured of coated for the @Epoxy coated and uncoatedparticles BIONs, overestimate determined the by actual dynamic particle diameter.light scattering This is (DLS) due at to pH still 10.-existing agglomeration effects, and liquid boundary layers, which surround the particles in the aqueous phase. A more accurate determination of the average size of a The charge of the particles can be explained by hydroxyl groups on the surface of the BIONs. single particle is possible by TEM [26]. These can be detected by FTIR spectroscopy. In the basic range, the Fe-OH group dissociates to form + Fe-O−. In the acidic range, a proton is taken up and Fe-OH2 is formed [29]. The stabilization of nanoparticles40 at alkaline pH values is necessary in order to obtain a homogeneous coating and thus a 16 BIONs 30 further functionalization with APTES. Stable non-agglomerated14 BIONs form the basis @Epoxy of the further 20 functionalization and antibody immobilization. APTES coated12 nanoparticles show a zeta potential of 10 17.59 at pH 6.4 and the @EpoxyPGA functionalized nanoparticles10 possess a zeta potential of 23.88 at 0 − pH 7.3, which further indicates a successful coating and functionalization.8 -10 Figure2b shows the DLS-measured size distribution of the BIONs6 at pH 10, where the nanoparticles -20 Zetapotential (mV) 4 are colloidally stable with aggregated structures of ~80 nmNumberdistribution (%) size and possess a high zeta potential -30 2

(>30 mV) [-4025]. 0 The silica2 coating4 6 and8 the10 polyglutaraldehyde12 activation10 stabilize100 the particles1000 against pH Hydrodynamic diameter (nm) agglomeration, which explains the smaller hydrodynamic diameters (~55 nm). The stabilization is caused by the mutual repulsion of electrostatically charged particles [28]. (a) (b)

Chemosensors 2020, 7, x FOR PEER REVIEW 6 of 12

Figure 2. (a) Zeta potential of BIONs at various pH-values titrated from pH 3 to pH 11 with sodium hydroxide (0.1 M). (b) Size distribution of coated @Epoxy and uncoated BIONs, determined by dynamic light scattering (DLS) at pH 10.

The functionality of a magnetic separation process is not only dependent on the functionalization but also on a high magnetization at elevated magnetic fields and superparamagneticChemosensors 2020, 8, 17 properties of the nanomaterials, which allow the manipulation in magnetic fields.6 of 12 Figure 3a shows the typical sigmoidal curves of superparamagnetic nanoparticles in a magnetic field. At an external field of 0 Oe the particles do not possess any remanence (<0.5 emu/g). Nevertheless, the values measured for the coated particles overestimate the actual particle diameter. BIONs reach the highest magnetization with 84.12 emu/g. This coincides with the results of This is due to still-existing agglomeration effects, and liquid boundary layers, which surround the previous measurements [30–32]. The APTES-coated particles reach up to 71.28 emu/g and the @Epoxy particles in the aqueous phase. A more accurate determination of the average size of a single particle is particles show a maximum of 68.93 emu/g. The decreasing magnetization is explained by the thin possible by TEM [26]. silicon layer on the particle surface after the coating process. A similar effect has been previously The functionality of a magnetic separation process is not only dependent on the functionalization reported by McCarthy et al. [32]. The crystal structure of the previously freeze-dried particle powder but also on a high magnetization at elevated magnetic fields and superparamagnetic properties of the was examined by XRD. The angles at which the reflections appear suggest the cubic face-centered nanomaterials, which allow the manipulation in magnetic fields. structure of crystalline magnetite. These can be assigned to the typical crystal planes designated in Figure3a shows the typical sigmoidal curves of superparamagnetic nanoparticles in a magnetic Figure 3b [33]. Typical maghemite reflections, such as (210) and (211), are not detected [34]. field. At an external field of 0 Oe the particles do not possess any remanence (<0.5 emu/g).

100 (311) 80 (440) 60 (511) (220) (400) BIONs (422) 40 (111) 20 0 @APTES

-20 Intensity (a.u.) Intensity -40 BIONs @Epoxy

Magnetization(emu/g) -60 @APTES -80 @Epoxy -100 -60000 -40000 -20000 0 20000 40000 60000 10 20 30 40 Field (Oe) 2q(°) (a) (b)

Figure 3. (a) SQUID-analysis of pure and coated BIONs at 300 K. (b) Powder X-ray diffractograms of Figure 3. (a) SQUID-analysis of pure and coated BIONs at 300 K. (b) Powder X-ray diffractograms of different nanoparticles. different nanoparticles. BIONs reach the highest magnetization with 84.12 emu/g. This coincides with the results of The surface properties of functionalized nanomaterials can be verified spectroscopically with previous measurements [30–32]. The APTES-coated particles reach up to 71.28 emu/g and the @Epoxy ATR IR spectroscopy. The prominent peak at about 600 cm−1 resembles a Fe-O vibration which is particles show a maximum of 68.93 emu/g. The decreasing magnetization is explained by the thin typical for Fe3O4 (Figure 4). Similar peaks are described by Ma et al. and Yamaura et al. [35,36]. Several silicon layer on the particle surface after the coating process. A similar effect has been previously small peaks between 700 and 1000 cm−1 can be attributed to Si-O vibrations. Examples are the band reported by McCarthy et al. [32]. The crystal structure of the previously freeze-dried particle powder of the Si-OH group at about 900 cm−1 or the band of the Si-O-Si bond between 900 cm−1 and 1000 cm−1 was examined by XRD. The angles at which the reflections appear suggest the cubic face-centered [35–38] The significant peaks at about 1550 cm−1 and 1635 cm−1 are attributed to the COO- group and structure of crystalline magnetite. These can be assigned to the typical crystal planes designated in the C=O bond, respectively. The appearance of these bands indicates a ring opening of the epoxy Figure3b [33]. Typical maghemite reflections, such as (210) and (211), are not detected [34]. rings. The characteristic bands of the C-O-C and C-O and C-C ring stretching modes at 1060 cm−1, The surface properties of functionalized nanomaterials can be verified spectroscopically with 1000 cm−1, and 900 cm−1 can be seen in the spectrum of @Epoxy particles, indicating a functional epoxy ATR IR spectroscopy. The prominent peak at about 600 cm 1 resembles a Fe-O vibration which is coating [29,37–39]. − typical for Fe O (Figure4). Similar peaks are described by Ma et al. and Yamaura et al. [ 35,36]. Considerin3 g4 the spectrum of the antibody-coupled particles in Figure 4, the great number of Several small peaks between 700 and 1000 cm 1 can be attributed to Si-O vibrations. Examples are −1 − −1 peaks in the frequency range between 800 cm and1 1200 cm is remarkable and verifies the complete1 the band of the Si-OH group at about 900 cm− or the band of the Si-O-Si bond between 900 cm− coupling of 0.2 mgAb/mgParticles. Absorbance measurements at 230 and 280 nm, and BCA assays of the and 1000 cm 1 [35–38] The significant peaks at about 1550 cm 1 and 1635 cm 1 are attributed to the supernatant −are in good agreement with IR spectroscopy data− , as we detected− no protein. The COO group and the C=O bond, respectively. The appearance of these bands indicates a ring opening antibody− load achieved for the @Epoxy+Ab particles are an improvement compared to previous of the epoxy rings. The characteristic bands of the C-O-C and C-O and C-C ring stretching modes studies [21,40]1 . Bloemen1 et al. [40] reach1 between 0.010 and 0.025 gAb/gParticles on magnetite particles at 1060 cm− , 1000 cm− , and 900 cm− can be seen in the spectrum of @Epoxy particles, indicating a functional epoxy coating [29,37–39].

Chemosensors 2020, 7, x FOR PEER REVIEW 7 of 12 with functional COOH groups loadings. The gold coatings of Zhang et al. [41] show a maximum loading of 0.088 gAb/gParticles which is similar to our results. These are characteristic oscillations of organic compounds, which essentially contain carbon, oxygen, and nitrogen. The peaks, which occur at about 1200 cm−1, can be ascribed to C-O and C-N bonds, and the bands of the C-O-C bond can be observed between 900 cm−1 and 1200 cm−1 [42,43]. The amide bands appearing at 1550 cm−1 and 1650 cm−1 indicate coupling [39]. Numerous bands are visible, which influence and overlap one another and make an exact assignment difficult. The spectrum of the Legionella used in this work is shown in Figure 4. Subsequently, it is compared with the spectrum of @Epoxy+Ab+Legionella complexes. Asymmetric and symmetrical stretching vibrations of the carboxyl group can be observed at about 1605 cm−1 and 1391 cm−1, respectively [44]. The first peak is enhanced by overlapping with the amide I band at 1635 cm−1. Other characteristic peaks are the amide II band at 1541 cm−1, which indicates polypeptide structures, and the methyl- diffraction band at 1456 cm−1. The same characteristic peaks at 1100 cm−1 and 1250 cm−1 appear in the

Chemosensorsspectrum 2020of pure, 8, 17 and particle-coupled Legionella samples and confirm a successful coupling process.7 of 12

@APTES

@Epoxy

@Epoxy+Ab

@Epoxy+Ab+Legionella Absorbance (a.u.) Absorbance

Legionella

3500 3000 1500 1000 Wave number (cm-1)

Figure 4. Attenuated total reflection infrared (ATR IR)-spectra of APTES-coated BIONs, @Epoxy nanoparticlesFigure 4. Attenuated with and total without reflection immobilized infrared antibodies. (ATR IR)- Thespectra spectra of APTES of particles-coated with BIONs, immobilized @Epoxy antibodiesnanoparticles is shown with beforeand without and after immobilized coupling with antibodies.Legionella Theand spectra the spectrum of particles of Legionella with immobilizedis shown. antibodies is shown before and after coupling with Legionella and the spectrum of Legionella is shown. Considering the spectrum of the antibody-coupled particles in Figure4, the great number of 1 1 peaksWe in the compare frequencyd the range immunomagnetic between 800 cm separation− and 1200 of cmantibody− is remarkable-coupled particles and verifies to BIONs. the complete Figure coupling5 shows a of decrease 0.2 mgAb of/mg theParticles optical. Absorbancedensity after measurementsseparation due atto 230bound and bacteria 280 nm,. At and c (BIONs BCA assays) = 0.4 ofg/L the, 75% supernatant of the starting are inOD good is still agreement present after with separation; IR spectroscopy whereas data, only 31% as we are detected present at no c protein.(BIONs) The= 16 antibody g/L. For loadlarger achieved concentrations, for the @Epoxy the polynomial+Ab particles approximated are an improvement curve flattens compared out. Thus, toprevious a further studiesincrease [21 of,40 the]. Bloemenparticle mass et al. hardly [40] reach influences between the 0.010 decrease and 0.025 in the g Aboptical/gParticles density.on magnetite particles with functionalThe optical COOH density groups of the suspension loadings. The after gold mag coatingsnetic separation of Zhang is etmainly al. [41 caused] show by a maximumthe present loadingcells. Turbidity of 0.088 gdueAb/ gtoParticles particleswhich is negligible is similar tobecause our results. of their almost complete magnetic separation, whichThese relates are to characteristic an OD of 0 for oscillations the particle of organicreference compounds,. The observed which separation essentially can containbe explained carbon, by 1 oxygen,the occurrence and nitrogen. of various The peaks, interactions which (van occur der at about Waals, 1200 electrostatic, cm− , can acid be ascribed-base) between to C-O and cells C-N and 1 1 bonds,particles and and the is bands therefore of the less C-O-C specific bond [22] can. be observed between 900 cm− and 1200 cm− [42,43]. 1 1 TheThe amide high separation bands appearing rate of at about 1550 70%cm− forand @Epoxy+Ab 1650 cm− indicate and BIONs coupling exceeds [39 ]. the Numerous results of bandspreviously are visible, performed which IMS influence experiments and overlap with E. one coli anotherfrom Tamer and makeet al. [22] an exact. The assignmentoptical density diffi ofcult. the Thesuspension, spectrum whichof the Legionella correspondsused to in this the work number is shown of Legionella in Figure 4 cells,. Subsequently, decreases it with is compared an increasing with thenanoparticle spectrum ofconcentration @Epoxy+Ab.+ TheLegionella opticalcomplexes. density decreases Asymmetric from and81% symmetrical when using stretchingthe lowest vibrations up to 30% 1 1 of the carboxyl group can be observed at about 1605 cm− and 1391 cm− , respectively [44]. The first 1 peak is enhanced by overlapping with the amide I band at 1635 cm− . Other characteristic peaks are 1 the amide II band at 1541 cm− , which indicates polypeptide structures, and the methyl-diffraction 1 1 1 band at 1456 cm− . The same characteristic peaks at 1100 cm− and 1250 cm− appear in the spectrum of pure and particle-coupled Legionella samples and confirm a successful coupling process. We compared the immunomagnetic separation of antibody-coupled particles to BIONs. Figure5 shows a decrease of the optical density after separation due to bound bacteria. At c (BIONs) = 0.4 g/L, 75% of the starting OD is still present after separation; whereas only 31% are present at c (BIONs) = 16 g/L. For larger concentrations, the polynomial approximated curve flattens out. Thus, a further increase of the particle mass hardly influences the decrease in the optical density. Chemosensors 2020, 7, x FOR PEER REVIEW 8 of 12

at the highest @Epoxy+Ab particle concentration (Figure 5). For BIONs, at an about ten times higher concentration of particles compared to @Epoxy+Ab, a similar decrease of the OD is achieved. The comparison with the separation by BIONs suggests that the interactions between antibodies and Legionella exceed those between pure particles and E. coli, as expected from the higher specificity of the binding [35]. In addition, the coated particles have smaller diameters and thus larger specific surface areas [45]. The achieved separation rate of 70% is in line with the achieved rates in previous IMS experiments or even surpasses them [22,46]. However, Füchslin achieved recovery rates of up to 92% using commercial Antibody-coupled MACS® microparticles from Miltenyi Biotec [19]. Since the same separation can be achieved by the addition of a larger number of particles without high-priced antibodies, it is preferable to use BIONs for non-specific separation. Chemosensors 2020, 8, 17 8 of 12

90 BIONs 80 @Epoxy+Ab 70

60

50

40 OD (%) OD 30

20

10

0

0.1 1 10 Particle concentration (g/L)

Figure 5. Optical density of the cell suspension after separation related to the density before. Separation byFigure BIONs 5.is comparedOptical density with separation of the cell by @Epoxysuspension+Ab-particle-complexes after separation related which to have the been density incubated before. withSeparation Legionella by (ODBIONs= 0.2 is compared corresponding with toseparation 100% or 5by @Epoxy+107 cells)Ab at- pHparticle 7.4 for-complexes 1 h. The which reference have × absorbancebeen incubated of water with incubated Legionella with (OD both = 0.2 nanoparticle corresponding species to 100% for all or particle5 × 107 cells) concentrations at pH 7.4 for (pH 1 h 7.4). The showsreference the same absorbance value as of water water after incubated magnetic with separation both nanoparticle of 1 h (OD species= 0). for all particle concentrations (pH 7.4) shows the same value as water after magnetic separation of 1 h (OD = 0). The optical density of the suspension after magnetic separation is mainly caused by the present cells. TurbidityDifferent duecell suspensions to particles is are negligible separated because by particle of their complexes almost completemade of @Epoxy magnetic and separation, Legionella- whichspecific relates antibodies. to an OD The of 0resulting for the particle opticalreference. densities are The shown observed in Figure separation 6a. A can considerable be explained difference by the occurrencebetween ofthe various Legionella interactions suspensions (van derand Waals, the other electrostatic, samples acid-base)is visible. betweenThen, 74% cells of and the particles cells are andseparated. is therefore In the less case specific of. E. [22 coli]. no separation is observed, which verifies the Legionella-specificity of @Epoxy+AbThe high separation complexes rate. of about 70% for @Epoxy+Ab and BIONs exceeds the results of previously performedThese IMS observations experiments are with consistentE. coli fromwith Tamerthe results et al. of [ 22Bloemen]. The optical et al. and density Füchslin of the et suspension,al., who were whichable to corresponds demonstrate to a the selective number separation of Legionella between cells, E. decreasescoli and Legionella with an increasing[19,21]. In Füchslin's nanoparticle IMS concentration.approach with The Legionella optical density-specific decreases antibodies from only 81% 1% when of the using present the E. lowest coli were up to separated 30% at the [19] highest. @EpoxyThe+Ab high particle separation concentration efficiency (Figure of even5). bare For BIONs, nanoparticles at an about was our ten timesmotivation higher to concentration investigate the ofbehavior particles compared of these nanoparticlesto @Epoxy+Ab, for a similar bacteria decrease separation. of the The OD is separation achieved. efficiency is strongly dependentThe comparison on the pH. with Ele thectrostatic separation interactions by BIONs between suggests the that particles the interactions and between between particles antibodies and cells andincrease Legionella with exceed their charge,those between which pure is consistent particles and withE. DLS coli, andas expected zeta potential from the data. higher An specificity improved ofcolloidal the binding stabilization [35]. In addition, leads to the a coated larger particleseffective havesurface smaller area diameters which is available and thus largerfor cell specific-particle surfacecoupling areas [44,46] [45].. TheThis achieved explains separationthe improved rate separation of 70% is inefficiency line with at the higher achieved pH-values rates in(Figure previous 6b). IMS experimentsThese results or even are quite surpasses promising them [ 22 for,46 the]. However, development Füchslin of a achieved process for recovery the concentration rates of up to of 92%Legionella using commercial pneumophila Antibody-coupled prior to detection MACS in water.®microparticles The time scale from with Miltenyi around Biotec 1 h of [19 the]. Sinceseparation the sameand separationconcentration can beprocess achieved is in by the the range addition of the of arequirements larger number for of clinical particles Legionella without tests high-priced in urine, antibodies, it is preferable to use BIONs for non-specific separation. Different cell suspensions are separated by particle complexes made of @Epoxy and Legionella-specific antibodies. The resulting optical densities are shown in Figure6a. A considerable difference between the Legionella suspensions and the other samples is visible. Then, 74% of the cells are separated. In the case of. E. coli no separation is observed, which verifies the Legionella-specificity of @Epoxy+Ab complexes. Chemosensors 2020, 7, x FOR PEER REVIEW 9 of 12 blood, and serum [5,6,47]. The process still demonstrates multiple challenges and the detection limit needs to be improved significantly with fluorescence labelling and accurate detection methods. In the future such a process can provide a sensitive cell enrichment method for Legionella detection, which is needed to control drinking water fast and cost-effectively in order to prevent the outbreak ofChemosensors Legionnaires’2020, 8 ,disease 17 in contaminated resources [48–50]. 9 of 12

120 80

100 60

80 (%)

60 40

OD (%) OD OD OD 40 20 20

0 0 Legionella E. coli MS no MS MS no MS MS no MS MS no MS 2.9 5.4 7.4 11.5 pH

(a) (b)

FigureFigure 6. 6. ((aa)) Optical Optical density density of the cellcell suspensionsuspension afterafter separation separation with with 0.8 0.8 g /g/LL @Epoxy @Epoxy++AbAb related related to the density before (OD 0.2 corresponding to 5 107 cells) at pH 7.4. The separation of different cell types to the density before (OD 0.2 corresponding to× 5 × 107 cells) at pH 7.4. The separation of different cell by Ab-particle-complexes was compared. The antibodies are specific to Legionella.(b) Optical density types by Ab-particle-complexes was compared. The antibodies are specific to Legionella. (b) Optical of the cell suspension after separation compared to the OD-value before separation (0.2 corresponding density of the cell suspension after separation compared to the OD-value before separation (0.2 to 5 107 cells) in %. The magnetic separation (MS) of Legionella with BIONs at different pH values corresponding× to 5 × 107 cells) in %. The magnetic separation (MS) of Legionella with BIONs at different is examined after incubation for 1 h. For comparison, samples without added BIONs for the same pH values is examined after incubation for 1 h. For comparison, samples without added BIONs for incubation time were also examined (no MS). the same incubation time were also examined (no MS). These observations are consistent with the results of Bloemen et al. and Füchslin et al., who were 4. Conclusions able to demonstrate a selective separation between E. coli and Legionella [19,21]. In Füchslin’s IMS approachWe were with ableLegionella to efficiently-specific antibodies couple Legionella only 1%-specific of the present antibodiesE. coli viawere an separated epoxy ring [19 opening]. reactionThe to high iron separation oxide nanoparticles. efficiency of The even successful bare nanoparticles coupling was of 0.2 our mg motivationAB/gParticle is to verified investigate by the IR spectroscopybehavior of these and nanoparticles further immunomagnetic for bacteria separation. separation. The With separation BIONs effi, ciency as well is asstrongly with dependentantibody- modifiedon the pH. magnetic Electrostatic nanoparticles interactions complexes, between the upparticles to 70 and% of between the present particles Legionella and cells cells increase can with be separated,their charge, which which corresponds is consistent to withthe results DLS and of previously zeta potential developed data. An IMS improved methods colloidal based on stabilization magnetic microparticlesleads to a larger [19,22,46] effective. surface About area 16 which mgParticles is available/mLcells of for BIONs cell-particle are needed coupling to [ 44 achieve,46]. This the explains same separationthe improved as with separation 0.8 mg@Epoxy+Ab efficiency/mL atcells higher of antibody pH-values-modified (Figure nanoparticles6b). . TheThese specificity results areof the quite antibody promising has great for theinfluence development on the success of a process of the separation for the concentration process. Only of withLegionella highly pneumophila specific antibodiesprior to detection is the IMS in method water. Thepreferable time scale to separation with around with 1 h pure of the particles separation, which and isconcentration much more processcost-efficient is in the. The range good of theperformance requirements of forpure clinical BIONsLegionella allows testsa comparatively in urine, blood, cheap and unsserumpecific [5,6 separation,47]. The process procedure still of demonstrates cells from liquids multiple (magnetic challenges separation, and the MS) detection without limit the needs use of to antibodies.be improved The significantly increasing with stabilization fluorescence of particle labelling suspensions and accurate in detection basic and methods. acidic environments In the future resultssuch a in process higher can separation provide arates. sensitive cell enrichment method for Legionella detection, which is needed to controlThus, drinkinga (selective) water pre fast-separation and cost-e withffectively magnetic in orderparticles to prevent and a subsequent the outbreak concentration of Legionnaires’ for detectiondisease in by contaminated fluorescence resourcesassays is suitable. [48–50]. Assuming a sufficiently sensitive analysis, the developed method represents a promising approach for the separation of Legionella from liquids. As shown by the4. Conclusionsexample of E. coli, the process can also be transferred to other cell types and thus offers a wide rangeWe of ap wereplications. able to e Otherfficiently (bio couple-) molecules,Legionella which-specific are antibodiesinteracting via with an the epoxy nanoparticles, ring opening could reaction be separated in the same way [12,22]. to iron oxide nanoparticles. The successful coupling of 0.2 mgAB/gParticle is verified by IR spectroscopy andMa furthergnetic immunomagnetic separation might separation. be a decisive With technology BIONs, as of well the asfuture with for antibody-modified bacterial enrichment magnetic and thereforenanoparticles for the complexes, faster detection up to 70% of bacteria. of the present Legionella cells can be separated, which corresponds Authorto the resultsContributions: of previously Conceptualization, developed IMS S.B., methods S.P.S., and based Y.K. onB.; magneticmethodology, microparticles J.A.T., F.S.; [validation,19,22,46]. AboutJ.A.T., F.S.,16 mg Y.K.ParticlesB., and/mL S.P.cellsS.; offormal BIONs analysis, are needed J.A.T.;to investigation, achieve the S. sameP.S., separationF.S., and J.A. asT.; with resources, 0.8 mg S.B.;@Epoxy data+Ab curation,/mLcells J.ofA. antibody-modifiedT.; writing—original nanoparticles. draft preparation, J.A.T. and S.P.S.; writing—review and editing, S.P.S. and S.B.; visualization,The specificity J.A.T. an ofd S. theP.S.; antibody supervision, has S.B great. and influence S.P.S.; project on the administration, success of the S.B.; separation funding acquisition, process. Only S.B. with highly specific antibodies is the IMS method preferable to separation with pure particles, which is much more cost-efficient. The good performance of pure BIONs allows a comparatively cheap unspecific separation procedure of cells from liquids (magnetic separation, MS) without the use of Chemosensors 2020, 8, 17 10 of 12 antibodies. The increasing stabilization of particle suspensions in basic and acidic environments results in higher separation rates. Thus, a (selective) pre-separation with magnetic particles and a subsequent concentration for detection by fluorescence assays is suitable. Assuming a sufficiently sensitive analysis, the developed method represents a promising approach for the separation of Legionella from liquids. As shown by the example of E. coli, the process can also be transferred to other cell types and thus offers a wide range of applications. Other (bio-) molecules, which are interacting with the nanoparticles, could be separated in the same way [12,22]. Magnetic separation might be a decisive technology of the future for bacterial enrichment and therefore for the faster detection of bacteria.

Author Contributions: Conceptualization, S.B., S.P.S., and Y.K.-B.; methodology, J.A.T., F.S.; validation, J.A.T., F.S., Y.K.-B., and S.P.S.; formal analysis, J.A.T.; investigation, S.P.S., F.S., and J.A.T.; resources, S.B.; data curation, J.A.T.; writing—original draft preparation, J.A.T. and S.P.S.; writing—review and editing, S.P.S. and S.B.; visualization, J.A.T. and S.P.S.; supervision, S.B. and S.P.S.; project administration, S.B.; funding acquisition, S.B. All authors have read and agreed to the published version of the manuscript. Funding: We appreciate support from the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open-Access Publishing Program and by TUM International Graduate School of Science and Engineering (IGSSE). The research was funded by the German Research Foundation (DFG) within the project 388673920. Acknowledgments: We thank BacTrace and specifically Walter Miedl, Andreas Eckelt and Behnam Kalali for providing antibodies and we would like to thank Carsten Peters for support with TEM imaging and Tom Nilges for the provision of the X-ray diffractometer. Furthermore, we want to acknowledge the work of Ramona Fischl, who performed preliminary experiments. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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