Magnetic beads Preparation Methods

Agarose coated magnetic beads

称取4g琼脂糖粉，加入40mL水，加热溶解，得到10%的琼脂糖溶液，降温至65 °C左右，向其中缓慢加入40%Na0H溶液0.5mL和双功能交联剂烯丙基缩水甘油醚6mL。其中，烯丙基缩水甘油醚在水相中的浓度为15%，0H—的浓度为0.21mol/L。在65°C下搅拌反应8h后，用60%的冰醋酸溶液调节pH值7〜8终止反应，得到了烯丙基化的琼脂糖溶液。向溶液中加入4倍体积的乙醇，离心收集沉淀，_70°C下预冻2小时，冷冻干燥72小时得到修饰的琼脂糖原料。

[0070] (2)共混制备交联剂修饰的琼脂糖微球

[0071] 准确称取修饰的琼脂糖原料1.20g (占共混原料总质量的50%)，未修饰琼脂糖原料1.20g (占共混原料总质量的50%)，共同加热溶解于60mL去离子水中，配成4%的共混琼脂糖溶液作为分散相。向600mL液体石蜡/石油醚的混合溶液中(V液体石蜡:V石油醚=11:1)，加入6%的油溶性乳化剂Span80，作为连续相。将油相预热至60°C，在自身重力作用下，分散相缓慢通过平均孔径25.9 μ m的疏水性SPG微孔膜进入到连续相中，形成均一的W/0型乳液。整个乳化过程在60°C下完成，制备期间用磁力搅拌使乳液保持均匀分散。膜乳化过程结束后，将乳液转入三口瓶中，在90rpm的搅拌转速下，将所得W/0型乳液缓慢降温至20°C以下，使其固化形成微球，并依次用石油醚、乙醇及蒸馏水洗涤得到浓度为4%的修饰琼脂糖微球。

[0072] ( 3 )琼脂糖微球内部交联

[0073] 将20g修饰琼脂糖微球分散于40g水中,加入2gNaAc,搅拌5min,滴加溴水至溶液呈深黄色，持续Imin不变色，继续反应15min。然后，加入甲酸钠至凝胶呈白色，水洗三次。将该溴化凝胶重新分散于20mL水中，加入2gNa2S04，搅拌15min后，加入40%Na0H溶液2mL(含有5%NaBH4)，反应2h。然后转入恒温水浴振荡器中，37.5°C振荡反应12h后，水洗至中性，得到浓度为4%的高交联琼脂糖微球。采用上述共混法制备的琼脂糖微球球形圆整(扫描电子显微镜如图1所示)，平均粒径85.33 μ m。

取7.5g琼脂糖于250mL水中(琼脂糖水溶液浓度为3%)，常温搅拌lh，加入

0.12gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.5L甲苯溶液，加入23.49g聚醋酸乙烯酯和2.349g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入22.5g环氧氯丙烷，分次滴加20mL50 %的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2°C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。

[0041 ] 实施例2

[0042] 取1g琼脂糖于250mL水中(琼脂糖水溶液浓度为4%)，常温搅拌lh，加入0.2gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.75L甲苯溶液，加入30.45g聚醋酸乙烯酯和3.045g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入30g环氧氯丙烷，分次滴加25mL50 %的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2 °C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。

[0043] 实施例3

[0044]取15g琼脂糖于250mL水中(琼脂糖水溶液浓度为6% )，常温搅拌lh，加入

0.375gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.625L甲苯溶液，加入35.3g聚醋酸乙烯酯和3.53g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入45g环氧氯丙烷，分次滴加38mL50 %的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2°C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。

[0045] 实施例4

[0046] 取20g琼脂糖于250mL水中(琼脂糖水溶液浓度为8% )，常温搅拌lh，加入0.36gKCl继续搅拌0.5h后，加热至琼脂糖沸腾，溶液呈现透明状。准备1.7L甲苯溶液，加入32.5g聚醋酸乙烯酯和3.25g油酸。将有机相于70°C下恒温搅拌至混合均匀，将沸腾的琼脂糖水溶液倒入有机相中，一定搅拌下琼脂糖的分散乳化lh。一次性加入60g环氧氯丙烷，分次滴加50mL50 %的NaOH溶液，Ih内滴加完毕。一定搅拌下固化3h后再进行冷凝，控制冷却速度2 °C/min至温度冷却至10°C以下。多次清洗后制备出粒径为100-300目的琼脂糖凝胶微球。性能测试

Method A: This method was containing two steps. In the first step, iron oxide nanoparticles were synthesized according to procedures reported in literature [22,23], in which Fe(II) and Fe(III) are co-precipitated in an alkaline solution. The particles were then separated, washed and dried in an oven. In the second step, the nanoparticles were added to a stirring 4% agarose solution at 80 ◦C. The solution was then cooled to 50 ◦C and a 4% (v/v) span85 in cyclohexane solution was added to the mixture under vigorous stirring [21]. Spherical agarose particles were produced containing several nano-magnetic cores.

Method B: It was a one-pot process in which preparation of iron oxide nanoparticles and their coating by agarose were performed simultaneously. For this purpose, common methods for the preparation of iron oxide nanoparticles were used [22,23], except that the alkaline solution was replaced by an alkaline agarose solution.

Briefly, 0.2 g FeCl3 and 0.073 g FeCl2 were dissolved in 1 mol L−1 HCL (12 mL). Then, 0.05 g agarose was dissolved in 2 mol L−1 NaOH (25 mL) under heating to 80 ◦C. The alkaline agarose solution was deoxygenated by purging nitrogen and 2 mL of the Fe3+/Fe2+ solution was added to it, dropwise. The addition was made under nitrogen atmosphere at reflux conditions (90 ◦C) and magnetic stirring (800 rpm). The color of the bulk solution changed to black immediately, that indicates the production of Fe3O4 nanoparticles. By this method, magnetic micro-flake agarose particles were produced. The final mixture was cooled to ambient temperature and the prepared ACMNPs were separated by a permanent magnet and washed with water and 50% (v/v) water/ethanol, respectively, and stored under 20% (v/v) ethanol in 4 ◦C. 2.4. Activation and functionalization procedures for ACMNPs For partial crosslinking of the agarose particles and their functionalization by IDA ligand, an epichlorohydrin activation method was used as reported elsewhere [24]. After activation, 1 mL of the ACMNPs was added to 10 mL 0.1 mol L−1 ligand solution (pH 10, adjusted by 1 mol L−1 NaOH). The mixture was then shaken for 24 h at 30 ◦C. Finally, the particles were washed well with water and stored as a 20% v/v suspension under 20% (v/v) ethanol in 4 ◦C for later uses. Fig. 2 shows an schematic representation of the preparation procedure for ACMNPs and their activation and functionalization by IDA.

Agarose Preparation methods

Preparation of a Fast-Flow Agarose-Based Chelating Adsorbent with a Novel Dioxime Derivative for Selective Column Preconcentration of Copper Payman HASHEMI,† Zohreh RAHMANI, Ali KAKANEJADIFARD, and Esmaiil NIKNAM

Preparation of spherical agarose particles Spherical homogeneous agarose particles (beads) of the particle size range (45 – 400 µm) were prepared according to a known emulsification method described by Gustavsson et al.18 A 20 ml solution of 6% (w/v) agarose was prepared by dissolution of the required amount of agarose powder in boiling water. The temperature was then adjusted on 60˚C in a water batch and 40 ml of cyclohexane containing span 85, 4% (v/v), with the same temperature, was added to the solution. The mixture was thoroughly stirred during the addition of cylohexane. After 3 min the mixture was cooled to room temperature by adding ice to the water bath. A cooling rate of about 5˚C/min was used. The spherical particles were then washed with ethanol and water and sized using graded metal sieves. Agarose particles with size ranges of 45 – 100, 100 – 200, and 200 – 400 µm were obtained in this way. Agar particles were prepared by simply grinding and sieving an agar powder (Merck). For increasing the rigidity of the particles, a patented method, described by Lindgren,13 was used with some modifications. Agarose (or agar) was first reacted with allylbromide in an alkaline medium, followed by a treatment with bromic water. In this way agarose was cross-linked in a defined way, and the number of side-reactions was minimized. This process was repeated until the desired rigidity was obtained.

https://patents.google.com/patent/CN105713212A/en

Embodiment 1

Taking 7.5g agarose (agarose solution concentration is 3%) in 250mL water, stirring at normal temperature 1h, after adding 0.12gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.5L toluene solution, add 23.49g polyvinyl acetate and 2.349g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 22.5g epoxychloropropane, the NaOH solution of gradation dropping 20mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.

Embodiment 2

Taking 10g agarose (agarose solution concentration is 4%) in 250mL water, stirring at normal temperature 1h, after adding 0.2gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.75L toluene solution, add 30.45g polyvinyl acetate and 3.045g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 30g epoxychloropropane, the NaOH solution of gradation dropping 25mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.

Embodiment 3

Taking 15g agarose (agarose solution concentration is 6%) in 250mL water, stirring at normal temperature 1h, after adding 0.375gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.625L toluene solution, add 35.3g polyvinyl acetate and 3.53g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 45g epoxychloropropane, the NaOH solution of gradation dropping 38mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.

Embodiment 4

Taking 20g agarose (agarose solution concentration is 8%) in 250mL water, stirring at normal temperature 1h, after adding 0.36gKCl continuation stirring 0.5h, heat and seethe with excitement to agarose, solution presents transparence.Prepare 1.7L toluene solution, add 32.5g polyvinyl acetate and 3.25g oleic acid.Organic facies constant temperature at 70 DEG C is stirred to mix homogeneously, the agarose solution of boiling is poured in organic facies, the dispersion and emulsion 1h of the lower agarose of certain stirring.Disposable addition 60g epoxychloropropane, the NaOH solution of gradation dropping 50mL50%, dropwise in 1h.Condense again after certain stirring lower solidification 3h, control 2 DEG C/min of rate of cooling and be cooled to less than 10 DEG C to temperature.Prepare, after repeatedly cleaning, the agarose gel microsphere that particle diameter is 100-300 order.

https://patents.google.com/patent/US3860573A/en

EXAMPLE 1

5 g of agarose or agar are suspended in benzene; n X 0.18 g triethylamine (n 1 are added. The reaction mixture is cooled close to the freezing point of benzene (about +6C) and n X 0.1 g oxalic acid chloride are added under powerful agitation. The temperature is allowed to rise up to +C (not higher) and the agitation is continued for another 6 hrs. The crosslinked agarose thus obtained is filtered and carefully washed with benzene, water and acetone and allowed to dry at room temperature.

EXAMPLE 2

5 g of agarose are suspended in ether; n X 0.14 triethylamine (n l 10) are added. The suspension is cooled to about +6C and thereafter n X 0.1 g maleic acid dichloride are added. Continuation as in Example 1.

EXAMPLE 3

5 g of agarose are suspended in petrol ether; n X 0.12 g N,N-dimethylaniline (n 1 10) are added. To the cooled (+6C) suspension n X 0.1 g cyclohexane l,2- dicarboxylic acid chloride are added. Continuation as in Example 1.

5 of agarose. are suspended in dioxane. In the mixture, n X 0.11 g (n'=1 10) of crystal water-free, recently dried sodium acetate are dissolved, and at about +10C, under agitation, n X 0.1 g succinic acid dichloride are added Continuation as in Example 1.

EXAMPLE 6 5 g of agarose are suspended in ether; n X 0.075 g triethylamine (n, 1 5) and n X 0.1 g benzene-1,3-.

disulphonic acid chloride are added. Reaction at +10 30C for 2-4 hrs is allowed. Continuation as in Example 1.

EXAMPLE 7 g of agarose are suspended in toluene; n X 0.1 g (n l-. 20) p-phenylene-diisothiocyanate are added. Under powerful agitation, the mixture is heated under a reflux cooler at the boiling point of toluene (+1 10C) for 8 hrs. Upon filtration, the cross-linked agarose thus obtained is washed with toluene and allowed to dry at room temperature.

EXAMPLE 8 5 g of agarose are suspended in dioxane; n X 0.1 g n l butyl-l,4-diisothiocyanate are added. Heating at the boiling point of dioxane (+l0lC) as in Example 7 is followed by washing with dioxane and acetone and drying at room temperature.

Cross-linked and epoxy activated agarose (Novarose) was a gift from Inovata AB (Stockholm, Sweden) with 40–60 �m particle size. Before use, Novarose was thoroughly washed with a 20ml portion of water, hydrochloric acid (1 mol l−1) and acetate buffer (0.1 mol l−1, pH 5.5), respectively

About 0.6 g of the ligand was dissolved in 40 ml 0.5 mol l−1 Na2CO3 and the solution’s pH was adjusted by 0.1 mol l−1 sodium hydroxide on 12.2. The solution was then added to a magnetically stirred suspension of 2ml of epoxy activated Novarose particles in 10 ml water and agitated for 24 h. The mixture was then filtered and the adsorbent was washed by 20 ml portions of 0.5 mol l−1 HCl, water, ethanol and water again, respectively. Deactivation of the remaining active sites was made by suspension of the adsorbent in 25 ml of a 1 mol l−1 sodium hydroxide solution and agitation for an extra 24 h. Finally, the adsorbent was washed by water and ethanol and stored at 4 ◦C in 20% ethanol.

Synthesis of Agarose

High-melting agarose D5 (Mr = 120 KDa, gelation temperature 46°-52° C, depending on concentration, composition and setting conditions, ρ(dry) = 1.64 g mL-1 ) (supplied by Millipore, UK) was used as raw material for the manufacturing of the adsorbent. White mineral oil Marcol N82 (ν(90° C) = 3.5 mm2s-1, ρ(90° C) = 812.3 kg m-3) (ESSO, UK) was used as the continuous phase. Sodium chloride (Sigma Aldrich, UK) was used as a co-solute for controlling the pore size of the adsorbent. Oil-soluble surfactants Span 80 (sorbitan monoleate, HLB 4.3, ρ(20° C) = 986 kg m-3, Mr = 446.62 g mol-1) and Span 85 (sorbitan trioleate, HLB 1.8, ρ(20°C) = 956 kg m-3, Mr = 975.51 g mol-1) (Sigma Aldrich, UK) were used for controlling particle size.

Agarose solution preparation Agarose powder at a concentration of 4 % (w/w) was dissolved in saline solutions of distilled water containing a certain amount of NaCl in a glass beaker. Distilled water was also added to account for water loss during subsequent heating. The mixture was then placed in a microwave oven for a total of 3 mins. To ensure complete dissolution of the agarose, the mixture was stirred by hand every 0.5 mins and for about 10 s. After obtaining a transparent solution the beaker was sealed and placed in a submersion water bath at 90° C where it was held for at least 30 mins to allow air bubbles to escape. Due to water evaporation a thick crust of dried agarose was formed on the surface of the solution. For this reason it was necessary to load the pre-heated syringe for the bottom of the beaker were the bulk concentration of the agarose solution remained constant. This was done by penetrating the crust of dried agarose with the syringe and immersing it into the beaker until it touched the bottom. Considering the viscous nature of agarose solutions, later observation of agarose beads with the SEM confirmed the mixing procedure followed resulted in homogenous mixture as the internal structure of the particles appeared fairly uniform. Emulsification A volume of 800 mL (80 % (v/v), continuous phase) of mineral oil was loaded to the emulsification vessel. Oil soluble surfactant of desired type and concentration was added to the vessel from the top port using a syringe. The mixture was allowed to equilibrate to 90° C while stirring at 1000 rpm for about 30 mins. Then, 4 L of mineral oil were placed in a plastic bottle and stored in deep freezer (-70°C) for approximately 45 mins thus cooling the oil to -10°C. After cooling the bulk volume of oil, 3.8 L were loaded to the quenching vessel and allowed to equilibrate for 30 mins to the desired quenching temperature while stirring at 300 rpm. As soon as the two vessels were equilibrated to the desired temperatures and air bubbles had escaped from the agarose solution, the high shear mixer rotor was fixed at the desired speed. Then, using a preheated syringe, 200 mL (20 % (v/v), dispersed phase) of the agarose solution were injected in the high shear mixer from the top port. After 5 mins of shearing the emulsion was discharged in the cooling vessel using compressed air (0.5 bars). To ensure complete gelation of the agarose particles, the mixture was allowed to equilibrate to the final cooling temperature (20° C) for at least 15 mins while stirring at 300 rpm. This relatively low speed was chosen in order to avoid altering the resulting particle size distribution coming out of the high shear mixer. The above concentrations and conditions were kept constant during the following experiments. To study the effect of ionic strength on pore size / size distribution and particle size / size distribution, NaCl was dissolved in the agarose solution at the following concentrations: 0, 0.01, 0.025, 0.05 and 0.1 molal. In this set of experiments the emulsion contained 0.1 % (v/v) Span 85, was emulsified at 30.7 W kg-1 (3500 rpm) and was quenched at 0° C. To study the effect of quenching temperature on pore size / size distribution and particle size / size distribution the emulsion was quenched at three different temperatures: 0°, 10° and 20° C, and three concentrations of salt were used at each quenching temperature (0, 0.01, 0.05 m NaCl), 0.1 % (v/v) Span 80 was added as a surface active agent. In all three experiments, formulations were emulsified at 30.7 W kg-1 (3500 rpm). To study the effect of surfactant concentration the resulting particle size / size distribution Span 85 was added to the continuous phase prior to emulsification at the following concentrations: 0, 0.1, 0.5, 1.0 and 1.35 % (v/v). The salt-free dispersion was emulsified at 2.87 W kg-1 (2000 rpm) and quenched at 0° C. The effect of surfactant type on particle size was studied by preparing two separate emulsions. In the first, Span 80 at a concentration of 0.1 % (v/v) was added to the continuous phase. In the second emulsion, Span 85 was added at the same concentration (0.1 % (v/v)). No salt was used in this experiment while the dispersions were emulsified at 30.7 W kg-1 (3500 rpm) and quenched at 0° C. Finally, to study the effect of rotor speed on the resulting particle size / size distribution two salt-free agarose solutions, each containing 0.1 % (v/v) Span 85, were emulsified at 2.87 (2000 rpm) and 30.7 W Kg-1 (3500 rpm) respectively , and quenched at 0° C.

Centrifugation

Following solid bead formation, the oil-particle mixture was transferred from the cooling vessel to the centrifuge (model Jouan C4 22, Thermo Fisher Scientific, UK). Centrifugation at 3000 rpm (1830 g) for 10 mins successfully separated the particles from the bulk volume of the oil as the liquid removed from the centrifuge bottles was transparent. A 25 % (v/v) aqueous ethanol solution was used to wash out the particles from the centrifuge bottles.

Washing

Prior to any characterization measurements particles where washed using the following procedure: A small volume of particles ( ̴30 ml) was placed in a sintered glass funnel connected to a vacuum pump. Particles where then washed with 3 volumes of 25 % (v/v) aqueous ethanol, 3 volumes of 50 % (v/v) ethanol, 3 volumes of 75 % (v/v) ethanol, 30 volumes of ethanol, 3 volumes of 75 % (v/v) ethanol, 3 volumes of 50 % (v/v) ethanol and finally 3 volumes of 25 % (v/v) ethanol. The successive washing with increasing following decreasing concentration of ethanol aqueous solutions prevents particles from possible structural damage caused by the rapid dehydration when washing directly with pure ethanol

(Amsterdam et al. 1975). After washing, particles were kept in 25 % (v/v) aqueous ethanol solution to prevent bacterial contamination.

Particle sizing

Particle size distributions, mean diameters (D(0,1), D(0,5), D(0,9) and D(3,2)), and yield (obtained from undersize curve) were determined with the Mastersizer 2000 (Malvern, UK). Refractive index values of 1.58 and 1.33 (Malvern manual) were used for agarose particles and distilled water respectively. The 25 % (v/v) aqueous ethanol solution was exchanged with distilled water prior to sizing. A 10 % (v/v) dispersion of particles was slowly added to the dispersion unit using a pipette until laser obscuration reached 10 % of its total value. A stirrer speed of 2000 rpm was used in the measuring cell (loop) to ensure complete dispersion of particles. Three measurements were performed per sample and the results presented in Chapter 3 are the average of said measurements.

Agarose Preparation Protocol 2

Preparation of Uniform-Sized Agarose Beads

A uniform sized agarose gel was prepared by emulsification technique, where 4% agarose was used as water phase and liquid paraffin was used as oil phase. Additionally, 2.5 wt% Tween 80 was added as surfactants which might help to prevent the coalescence of agarose gel. So agarose was dissolved in distilled water with containing 0.9% NaCl and the solution was heated by microoven to a temperature above the gelation point of the agarose. The solution was designated as water phase. The mixture of liquid paraffin and Tween 80 was heated to a temperature above the gelation point of the agarose approximately 80°C and the solution was designated as oil phase. The oil phase was charged into the water phase and agitated quickly by agitator. A uniform sized emulsion was formed in the reactor. The emulsion was added into second hydrophobic liquid cooled paraffin at -20°C to solidify the agarose droplets into beads. Finally the agarose beads were recovered from the second liquid and washed with distilled water. After washing the gel was screened, standardized mesh No. series as 25, 60, 120, and 270 was used to determine the particle size distribution of a granular material. After screening, the obtained uniform size gel was sent for microscopic view.

Cross-Linking of Agarose Gel by Epichlorohydrin

About 50 ml uniform-sized agarose gels were taken in a 250 ml beaker containing 20 ml of 2 M NaOH and 5ml of Epichlorohydrin. The mixture was allowed to react for 2 hours at 40°C with constant mixing in a home made rotary mixture to keep the concentration uniform [17]. After reaction, the gels were washed with deionized distilled water until it became neutral (conforming by litmus paper). 2.3. Modification of Agarose Gel with 1,6-Hexanediamine For the modification of agarose gel, 300 ml 0.2 M of 1, 6 hexadiamine solution containing 1.5 gm sodium carbonate decahydrate was mixed with the epoxy cross-linked agarose gels. The reaction was carried out at 60°C for 2 hours [18]. After reaction the gels were again washed with distilled water until the gel became neutral.

Activation of Agarose Gel with 2,4,6-trichloro-1,3,5-trizine

Amino modified agarose gel was soaked in 50 ml of 3 M NaOH solution. It was placed in ice bath and 150 ml of a 0.5 M 2,4,6-trichloro-1,3,5-trizine solution was mixed with homemade mixture in refrigerator for 60 mins. After activation, the gels were rinsed successively with 500 ml of 50 vol% acetone aqueous solution and 2 liter distilled water was used for washing to remove the unreacted 2,4,6- trichloro-1,3,5-trizine.

Immobilization of ConA to the 2,4,6-trichloro-1,3,5-trizine

Activated Gel 2,4,6-trichloro-1, 3, 5-trizine activated gel(collected from title 2.4.) was placed into an ice bath and mixed with 0.1 M sodium phosphate buffer, pH 7.8 containing 200 mg of commercially conA with continuos mixing for 12 hrs. After coupling, the gel was separated and 150 ml of a 0.5 M ethanolamine aqueous solution was mixed for 8 hrs to block the unreacted triazine groups. Finally the gels were rinsed with 1 liter distilled water [19]

Cross-linked agarose 

http://www.jbc.org/content/254/8/2572.full.pdf

A Novel Method of Activation of Cross-linked Agaroses with 1,l carbonyldiimidazole Which Gives a Matrix for Affinity Chromatography Devoid of Additional Charged Groups* 

Cross-linked agarose (Sepharose CL-GB) was purchased from Pharmacia (Uppsala, Sweden), 1,1’-carbonyldiimidazole from Pierce (Rockford, Ill.), and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate from Merck (Rahway, N. J.). All other chemicals were of reagent grade and were used without further purification. Dioxane contained not more than 0.5% water. Subsequent to the completion of these studies, Pierce Chemical Co. has agreed to make the activated matrix commercially available as “CDI’-activated Agarose.” Trinitrobenzene sulfonic acid was purchased from Aldrich (Milwaukee, Wise.). Typical Activation Procedure-Sepharose CL-6B (3 g of moist cake) was washed sequentially with water, dioxane/water, 3:7; dioxane/water, 7:3; and dioxane (20 ml of each) and was suspended in dioxane (5 ml). CD1 (0.12 g) was added, and the suspension was shaken at room temperature for 15 min. It was washed with dioxane (100 ml) and used immediately. Coupling of Amines-The above material was treated at 4°C overnight with n-butylamine, 1,6-diaminohexane, or 6-aminohexanoic acid (1.4 g) in water (9 ml) at pH 10 and washed sequentially with water (200 ml), 1 M NaCl (100 ml), and water (200 ml). Materials for titration were further washed with 5 mu HCl (200 ml). Coupling of Ethanolamine to 6-Aminohexanoic Acid-SepharoseThe 6-aminohexanoic acid-Sepharose CL-6B (3 g of moist cake) was treated at room temperature with ethanolamine (0.5 g) and CMC (0.5 g), and the pH was adjusted to 6 and maintained overnight at that pH with a Radiometer pH titrator (TTTB). The final volume was 12 ml. The material was then washed as for the coupling step. Affinity Chromatography-Affinity chromatography was carried out on 0.4- to l-ml samples packed into Pasteur pipettes. The equilibrating, loading, and washing buffer was 0.05 M Tris (pH 8) and desorption was carried out with 3 mM HCl. All solutions contained 0.5 M NaCl. Loadings were 20 mg of trypsin in 16 ml of buffer for A, B, and D and 10 mg in 8 ml for C.

Silica-Coated Magnetic Nanoparticles

A magnetic fluid with particles with a size of 15 nm are prepared by using conventional coprecipitation of Fe3+ and Fe2+ ions by OH^-1 at room temperature. In a typical synthesis, 4.17 g of FeCl3.6H2O and 1.52 g of FeCl2.4H2O (such that Fe3+/Fe2+ = 2) are dissolved in 80 ml of water (concentration of Fe2+ is 0.1 M) with vigorous stirring. A solution of 6 ml of 35 % NH4OH is added at a rate of 1 drop per second at room temperature during constant stirring. Black precipitates of Fe3O4 (FeO.Fe2O3) are formed and isolated from the sol-vent by magnetic decantation. The water washing and the decantation process are repeated four times to remove excess solution. In a similar way, Fe_1~xNi _xO.Fe2O3 and Fe_1~yCo_y.Fe2O3 nanoparticles with x = 0.05, 0.1, 0.2, 0.5 and y = 0.2, 0.4 are made by using NiCl2.6H2O and CoCl2.6H2O, respectively to replace Fe2+ by Ni2+ and Co2+. All procedures are conducted under a N2 atmosphere. To study the oxidation of bare NPs, after washing, we dried the FeO.Fe2O3, Fe_1~xNi_xO.Fe2O3 and Fe_1~yCo_y.Fe2O3 nanoparticles and exposed them in air for them to be oxidized; then we measured the magnetic properties. To see the effect of surfactant coating, we vigorously mixed the precipitates in water with polyisobutylene-modi ed-polysuccinimide (PIB-PS) and
then redisperse them in hexane as the carrier liquid. The hexane washing and the decantation are conducted four times to remove excess PIB-PS in the solution. After
that, the PIB-PS-coated nanoparticles  are dried and exposed in air for them to be oxidized.

Synthesis of Silica-Coated Magnetic Nanoparticles

https://www.hindawi.com/journals/jnm/2013/603940/

Magnetic Fe3O4 nanoparticles were synthesized by using coprecipitation from iron (III) chloride and iron (II) chloride solutions with the assistance of aqueous ammonia solution as described elsewhere [6]. The synthesized magnetic nanoparticles were washed several times with alcohol and then distilled water until pH 7.0. Fe3O4/SiO2 nanoparticles were prepared by coating magnetic nanoparticles with silica. 100 mL of the suspension of prepared magnetic nanoparticles (containing 1 g magnetite) was stored in a flask. 50 mL of the 10% solution of aqueous tetraethylorthosilicate (TEOS) was added to the flask together with 70 mL alcohol and mixed using an overhead stirrer. The pH of the suspension was adjusted to 9.0 with NaOH. The flask was then heated to 90°C and stirred at this temperature for 6 h. After cooling to room temperature (RT), the suspension was washed twice with alcohol and six times with distilled water. The final suspension volume was adjusted to 100 mL with water.

Fe3O4/SiO2 nanoparticles were characterized using a transmission electron microscope (TEM JEM1010, JEOL) and Fourier-transform infrared (FTIR) spectroscopy (FT/IR-6300, JASCO). Magnetic curves were measured using a DMS-880 vibrating sample magnetometer (VSM) at RT.

1. 0.02~0.42 mole of FeCl3 and 0.02~0.3 mole of FeCl2 in 18~500 ml water was heated at 60~90° C. for 10~30 minutes to obtain a mixture.

2. The above mixture was mixed with 5~25% of alkaline solution and stirred at 60~90° C. for 20~45 minutes to form magnetic cores.

3. Cooled the magnetic cores to room temperature. 4. Alkaline solution (0.05X ~ 0.1X volumes) and ethanol (1X ~3 X volumes) were then mixed to step 3.

5. Tetraethyl orthosilicate (TEOS) was added to step 4 in various ratio (0.05%~ %) to coat the magnetic cores with silica forming silica-coated magnetic beads.

6. The silica-coated magnetic beds were incubated overnight at room temperature.

7. The silica-coated magnetic beads were washed sequentially by ethanol and ddH2O.

8. The silica-coated magnetic beads were finally stored in ddH2O at room temperature.

DNA isolation

For further experiments in nucleic acid isolation, Fe3O4/SiO2 nanoparticles were stored in 100 mM Tris-HCl buffer (pH 7.0). For each DNA isolation reaction from 200 μL serum sample, 50 μL of Fe3O4/SiO2 nanoparticles at a concentration of 25 mg/mL was used.

2.2. Preparation of DNA Isolation Buffers A set of nucleic acid buffers were made based on the method described previously by Boom et al. [1] and optimized in this work suitably for properties of Fe3O4/SiO2 nanoparticles. The set contained (i) Proteinase K 20 mg/mL (BioBasic), (ii) lysis buffer (LB: 30 mM Tris-HCl pH 7.0, 1 M NaCl, 4.5 M GuSCN, 20 mM EDTA, 1.5% Triton X-100), (iii) two types of washing buffers (WB1: 30 mM Tris-HCl, pH 7.0, 2.25 M GuSCN, 56% ethanol; WB2: 10 mM Tris-HCl pH 7.0, 70% ethanol), and (iv) elution buffer (EB: 10 mM Tris-HCl pH 8.5, 1 mM EDTA).

three ways to protect nanoparticles from oxidation: (a) using a highly viscous carrier liquid, which limits the dilusion of oxygen; (b) using relevant surfactants, PIB-graft-PS and PIB-block-PS; (c) substitution of Ni2+ and Co2+ for Fe2+ in the structure of magnetite. In (b) the hydrophobic tail (PIB) should be a saturated, straight hydrocarbon chain and the hydrophobic head (PS) should be amine group.

Silica coated superparamagnetic microbead

https://www.hindawi.com/journals/jnm/2016/1730403/

Tetraethylorthosilicate (TEOS), ammonium hydroxide (NH4OH), styrene, 2,2′-azobis-isobutyronitrile (AIBN), dibenzoyl peroxide (BPO, including 97% of active compound), dibutyl phthalate (DBP), sodium silicate, Fe3Cl3·6H2O, FeCl2·4H2O, ethylene glycol, N,N-dimethylformamide (DMF), sodium dodecyl sulfate (SDS), polyvinylpyrrolidone-40 (PVP-40, Mr: 40,000), N,N-diisopropylethylamine (DIEA), and (3-aminopropyl)triethoxysilane (APTS) were purchased from Sigma-Aldrich (MO, USA).

2.2. Methods 2.2.1. Preparation of Monodisperse Macroporous PS-DVB Beads Monodisperse macroporous PS-DVB (polystyrene-divinylbenzene) beads were prepared by seeded polymerization methods. Monodisperse PS seeds (4 μm) were prepared using dispersion polymerization method [49]. Dispersion medium was ethanol/2-methoxyethanol (3 : 2) which contains 1 g of PVP-40 as a steric stabilizer (90 mL). AIBN (150 mg) was dissolved in styrene (15 mL) in which inhibitor was removed and then was added to the as-prepared dispersion medium. After sonication for 10 min, dispersion polymerization was performed in a cylindrical reaction chamber with shaking (120 cpm) at 70°C for 20 h. The suspension was centrifuged and the precipitates were washed intensively with distilled water by repeating centrifugation and vortexing. Finally, the PS seeds were washed with ethanol and vacuum-dried overnight affording PS seeds (4 μm, 8.3 g).

Swelling and polymerization processes were performed in a glass reactor equipped with overhead stirrer and a reflux condenser in an oil bath equipped with a temperature controller. The two-step seeded polymerization method was employed for preparation of monodisperse macroporous poly(styrene-co-DVB) (PS-DVB) beads. The PS seeds (4 μm, 700 mg) were dispersed in DBP (0.7 mL) emulsified aqueous medium (100 mL) containing 0.25% (w/w) SDS. The resultant dispersion medium was stirred (400 rpm) at room temperature for 20 h for swelling the PS seeds with DBP. Mixture of styrene (4.6 mL) and DVB (2.3 mL) in which BPO (240 mg) was dissolved was emulsified in an aqueous medium (100 mL) containing 0.25% (w/w) SDS by using homogenizer for 1 min. The emulsified monomer solution was added to the DBP-swollen PS seeds dispersion medium being stirred. Monomer swelling was performed for 20 h at room temperature with continuous stirring (400 rpm). After swelling process, an aqueous solution of 10% (w/v) PVA in deionized water (10 mL) was added to the dispersion medium and the medium was purged with nitrogen stream for 30 min. The seeded polymerization was performed at 70°C for 20 h with continuous stirring (200 rpm) to obtain monodisperse PS-DVB beads. The polymer beads were intensively washed with warm deionized water (50°C) by repeating washing and centrifugation. Subsequently, the collected beads were washed with ethanol and THF intensively to remove DBP and linear polymer. Finally, the beads were dried in vacuum at 30°C for 24 h to obtain macroporous PS-DVB beads (7.5 μm, 2.5 g).

2.2.2. Sulfonation of Macroporous PS-DVB Beads Sulfonation of the PS-DVB beads was performed with reference to the reported method [50]. Briefly, monodisperse PS-DVB beads (1 g) were added to 5 mL of acetic acid in an ice bath. Sulfuric acid (50 mL) was then slowly added to the beads at room temperature and the temperature was increased up to 90°C and the resin mixture was stirred for 30 min to 2 hr. The dispersion was poured into iced water (400 mL) to quench the reaction and the sulfonated PS-DVB beads were collected by centrifugation. The beads were extensively washed with deionized water by repeating centrifugation-decantation. Subsequently, the sulfonated microspheres were washed three times with ethanol and dried under vacuum (1.1 g).

2.2.3. Magnetization of Sulfonated Macroporous PS-DVB Beads The sulfonated macroporous PS-DVB beads (500 mg) were dispersed in deionized water (10 mL) at room temperature with mechanical stirring (200 rpm) and purging with nitrogen. A freshly prepared mixture of FeCl3·6H2O (618 mg, 2.26 mmol) and FeCl2·4H2O (257 mg, 1.28 mmol) in deionized water (10 mL) was added to the dispersion for adsorption. After 2 h, with continuous stirring 28% ammonium hydroxide (50 mL) was added dropwise to the beads suspension for 40 min. The magnetized microspheres were isolated from the mixture by centrifugation and briefly washed with 25% TFA and then extensively with deionized water and ethanol. Finally, the magnetized microspheres were dried under vacuum (magnetized microspheres, 7.5 μm, 653 mg).

2.2.4. Preparation of Silica Coated Magnetic Beads APTS solution (1% (v/v), 100 mL) was added to 100 mg of magnetic sulfonated PS-DVB beads and was shaken for 10 min at room temperature. Thereafter, ammonium hydroxide (28%, 2 mL) was added to the bead dispersion and was shaken for 20 min at room temperature. To the dispersion TEOS (2 mL) was added and vigorously shaken for 12 h at room temperature. The resulting beads (silica coated magnetic beads) were collected by magnet and washed with ethanol five times.

2.2.5. Streptavidin Separation of Silica Coated Magnetic Beads APTS (240 μL) and ammonium hydroxide (40 μL) were added to 4 mL absolute ethanol containing 200 mg of silica coated magnetic beads and incubated for 6 h at room temperature. The resulting beads (amine-silica coated magnetic beads) were collected by magnet, washed with ethanol five times, and dispersed into PBS, pH 7.2, to obtain 10 mg/ml of amine-silica coated magnetic beads.

Biotin (5 mg) was activated with EDC (40 mg) and NHS (20 mg) in PBS, pH 7.2, for 1 h at room temperature. Then, this solution was added into amine-silica coated magnetic beads (1 mg, 100 μL) to modify biotin on its surface. The biotinylated silica coated magnetic beads were collected by magnet, washed with ethanol five times, and dispersed into 100 μL PBS, pH 9.0. The biotinylated silica coated magnetic beads were passivated with 5% BSA in PBS, pH 9.0, for preventing nonspecific binding.

Streptavidin-FITC (100 μg) in PBS pH 9.0 was added into the biotinylated silica coated magnetic beads and incubated for 30 mins at room temperature. The biotinylated silica coated magnetic beads after incubation with streptavidin-FITC were collected by magnet, washed with PBS, pH 9.0, containing 0.1% Tween 20 for five times, and dispersed into 100 μL PBS, pH 9.0. The fluorescent images were collected by the Olympus BX-UCB at room temperature with 200 ms exposure.

Polymer Brush-Modified Magnetic Nanoparticles

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153590/

Sigma: N,N-dimethylformamide (DMF, anhydrous, 99.8%), CuCl (99.999%), CuBr2 (99%), 2,2’-bipyridyl (bpy, 99%), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), imidazole (99%), 2-hydroxyethyl methacrylate (HEMA, 97%), TWEEN-20 surfactant

HEMA was passed through a column of activated basic alumina (Aldrich) prior to use. NiSO4·5H2O (Columbus Chemical), CuSO4·5H2O (CCI), NaH2PO4 (CCI), Na2HPO4 (Aldrich), tetraethyl orthosilicate (TEOS, Fluka), Nα,Nα-bis(carboxymethyl)-l-lysine hydrate (aminobutyl NTA, Fluka), succinic anhydride (SA, Matheson Coleman & Bell),

https://ferrofluid.ferrotec.com/products/ferrofluid-emg/water/emg-308/

EMG 308 (1 mL, Ferrotec), a solution of Fe3O4 nanoparticles, was diluted with water (13.6 mL) prior to addition of isopropanol (80 mL) to precipitate the magnetite nanoparticles and remove excess surfactant. The supernatant was decanted, and the magnetically collected nanoparticles were then dispersed in 200 mL of 80% (v:v) ethanol (EtOH) in water. After sonication of this solution for 15 min, concentrated ammonia (5 mL) and TEOS (2 mL) were added to the solution,37, 38 and the mixture was mechanically stirred for 3 h. HCl (1 mL, 2M) was added to stop the reaction, and the silica-coated MNPs were magnetically collected and washed twice with 50 mL of EtOH (the same volume of solvent was used for all the washing steps), twice with water, and again with EtOH before drying under vacuum for 6 h.

Initiator attachment

SiO2-MNPs were dried at 120 °C overnight and then dispersed in toluene through sonication (0.2 g beads in 150 mL). Tricholorosilane initiator, (11-(2-bromo-2-methyl) propionyloxy)undecyltrichlorosilane39 was added (0.2 mL), and the mixture was stirred at 65 °C overnight while bubbling with N2. The initiator-modified beads were washed twice with 50 mL of toluene and twice with 50 mL of THF prior to drying in vacuum for 4 h.

(11-(2-bromo-2-methyl) propionyloxy)undecyltrichlorosilane

tethered initiator for atrp on silicon surfaces.

2-Hydroxyethyl methacrylate:  500 G $33.90 https://www.sigmaaldrich.com/catalog/product/aldrich/128635

2,2′-Bipyridyl

DMAP

https://www.sigmaaldrich.com/catalog/substance/4dimethylaminopyridine12217112258311

Growth of poly(2-hydroxyethyl methacrylate) (PHEMA) on beads

Initiator-modified beads (0.1 g) were dispersed by sonication in purified HEMA (10 mL), and the mixture was degassed via 3 freeze-pump-thaw cycles. In another Schlenk flask, HEMA (10 mL) was mixed with water (10 mL) and bpy (366 mg), and after 3 degassing cycles CuCl (82.5 mg) and CuBr2 (54 mg) were added and another 2 degassing cycles were performed. In a glove bag, the two degassed HEMA mixtures were combined and magnetically stirred for 1 h. Subsequently, air was bubbled through the polymerization solution until its color turned from dark brown to dark green, and the beads were then washed sequentially with 20 mL of DMF (the same volume of solvent was used for all the washing steps), EtOH (twice), water, and EtOH, and dried in vacuum.

Derivatization

40 PHEMA-MNPs (200 mg) were dispersed in 10 mL DMF by sonication and mixed with 10 mL DMF containing SA (200 mg) and DMAP (200 mg) prior to stirring at 55 °C overnight. The SA-MNPs were washed with 20 mL of DMF (the same volume of solvent was used for all the washing steps), EtOH (twice) and then water (twice). Subsequently, an aqueous solution (20 mL) containing EDC (0.1 M) and NHS (0.1 M) was mixed with the SA-MNPs for 30 min followed by washing with EtOH three times. Then, the EDC/NHS-activated beads were exposed to a pH 10, aminobutyl-NTA (0.1 M) aqueous solution for 1 h with magnetic stirring and washed with water three times. Finally, the NTA-SA-PHEMA-MNPs were immersed in CuSO4 (0.05 M) for 1 h, washed with water three times and EtOH twice and dried under vacuum for 5 h.

Preparation of NiFe2O4 Nanoparticles

NiFe2O4 NPCs were synthesized by a hydrothermal process using nickel chloride hexahydrate (NiCl2$6H2O, ReagentPlus, Sigma-Aldrich) and iron chloride hexahydrate (FeCl3$6H2O, 97.0%, Samchun Chemicals) as the precursors.17,21,22 Ethylene glycol (99.5%, Samchun Chemicals) and sodium acetate (anhydrous, 99.0%, Sigma-Aldrich) were used for the formation of NPCs in this polyol process. The detailed roles of these reactants have been described in previous reports.21,23,24 First, 0.36 g (1.5 mmol) of NiCl2.6H2O and 0.81 g (3.0 mmol) of FeCl3.6H2O were dissolved in 30 mL of ethylene glycol and stirred until a clear solution was formed. Then, 2.16 g of sodium acetate was added to it with vigorous stirring. After 30 min, a KNO3 solution (0.30 g of KNO3 in 2 mL of distilled water) was added and stirred for 10 min. The obtained solution was transferred to a Teflonlined stainless steel autoclave (100 mL capacity) and sealed. The autoclave was heated at 200 oC for 8 h and then naturally cooled for 30 min followed by forced cooling to room temperature (25 oC) in a flowing water bath. The obtained particles were washed several times with ethanol, and then dried at 60 oC for 10 h. spherical NPCs with diameters of approximately 100 nm. The result of dynamic light scattering analysis shows that the average size of NPCs in aqueous dispersion was ~150 (~45) nm.

Binding: 50 mM  Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0 
buffer I: 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, and pH 8.0  
Wash buffer II: 50 mM Tris-HCl, 300 mM NaCl, 40 mM imidazole, and pH 8.0 
Elution buffer: 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, and pH 8.0 for 10 min, recovery: (60%) were recovered
Reclycling: can reuse 7 times
Capacity: 100 mg of Histagged sGFP can bind to 1 g of NiFe2O4 NPCs

Briefly, 0.2 g FeCl3 and 0.073 g FeCl2 were dissolved in 1 mol L−1 HCL (12 mL). Then, 0.05 g agarose was dissolved in 2 mol L−1 NaOH (25 mL) under heating to 80 ◦C. The alkaline agarose solution was deoxygenated by purging nitrogen and 2 mL of the Fe3+/Fe2+ solution was added to it, dropwise. The addition was made under nitrogen atmosphere at reflux conditions (90 ◦C) and magnetic stirring (800 rpm). The color of the bulk solution changed to black immediately, that indicates the production of Fe3O4 nanoparticles. By this method, magnetic micro-flake agarose particles were produced. The final mixture was cooled to ambient temperature and the prepared ACMNPs were separated by a permanent magnet and washed with water and 50% (v/v) water/ethanol, respectively, and stored under 20% (v/v) ethanol in 4 ◦C. 2.4. Activation and functionalization procedures for ACMNPs For partial crosslinking of the agarose particles and their functionalization by IDA ligand, an epichlorohydrin activation method was used as reported elsewhere [24]. After activation, 1 mL of the ACMNPs was added to 10 mL 0.1 mol L−1 ligand solution (pH 10, adjusted by 1 mol L−1 NaOH). The mixture was then shaken for 24 h at 30 ◦C. Finally, the particles were washed well with water and stored as a 20% v/v suspension under 20% (v/v) ethanol in 4 ◦C for later uses. Fig. 2 shows an schematic representation of the preparation procedure for ACMNPs and their activation and functionalization by IDA.

Magnetic Colloidal Cube (Protocol from Youtube.)

56g Iron Chloride Hexahydrate --->250 ml pyrex

add H2O to 100 ml to dissolve

Slowly add 90 ml of 6 M NaOH while stirring

Add 10 ml ddH2O

Silica Coated Magnetitite   (Protocol from Youtube.)

2:1 ratio Fe³⁺: Fe_²⁺, 2.5 g FeSO4-40 ml H2O

140 ml ammonium, slowly , 1.5 hours, orange --> black. until pH is 10.

use sodium silicate  Sodium silicate | Na2SiO3 to coat the particle

Polydopamine Modified Glutathione disulfide Superparamagnetic IronOxide Nanoparticles

https://www.ncbi.nlm.nih.gov/pubmed/30678236 https://www.mdpi.com/2079-4991/9/2/138

Iron oxide nanoparticles (Fe3O4, IONPs) were prepared by a facile chemical co-precipitationmethod [21] where FeCl3·6H2O (34.6 g) and FeCl2·4H2O (12.7 g), in the molar ratio of 2:1,were dissolved in 1.5 L of water. Then, Fe3O4 nanoparticles were precipitated quickly by addingammonia (120 mL, 28%) in the solution at 22 ◦C. The obtained black suspension was stirred for 2–3 min.The obtained black precipitate was then magnetically decanted and washed thoroughly with deionized

(DI) water until the solution reached pH 8. The precipitate was then kept in nitric acid solution (1 mM,pH 3) and dialyzed against nitric acid solution (10 mM) for 48 h. Dialyzed nanoparticles were thencentrifuged for 15 min at 20,000 G with deceleration to obtain stable IONPs. The supernatant was thenused for further characterizations and surface modification Synthesis of Polydopamine-modified IONPs: pDA-IONPsA quantity of 100 mg of Fe3O4 (10 mg mL−1) was dispersed under continuous stirring in 25 mL of10 mM DA solution (PBS, 10 mM, pH 9.0) for 24 h at 22 ◦C. After, polydopamine-modified nanoparticles(pDA-IONPs) were magnetically decanted, washed thoroughly 5 times with ultrapure water using ultrafiltration to remove the non-reacted DA (ensured using UV), and further dispersed in water.

Preparation of GSSG-modified Nanoparticles

GSSG-modified nanoparticles were prepared using 20 mg of pDA-IONPs, which were dispersed in 1 mg mL􀀀1 GSSG solution (10mMPBS, pH 9.4) for 24 h under continuous magnetic agitation (700 rpm) at 22 �C. The GSSG-modified nanoparticles (GSSG-pDA-IONPs) were magnetically separated using magnetic syringe [22], washed with PBS buffer and checked with DTT to ensure the complete removal of unreacted GSSG. DTT reduces the unreacted disulfide, giving a yellow color solution indicating the presence of disulfide group. The resultant nanoparticles were then washed and re-dispersed in PBS. The above nanoparticles were then reduced to glutathione to get free –SH. Briefly, GSSG modified nanoparticles (800 �L of the suspension) were first mixed with Tris buffer (0.2 M, pH 9), after which, DTT (0.01 M) was added and the reduction of disulfide was allowed to proceed for 1 h at 22 �C. To ensure the reduction, DTNB (0.01 M) in acetate buffer (0.2 M, pH 5) was reacted with the sample and the absorbance was measured at 408 nm. S-nitrosation was then performed using NaNO2 (0.4 mM). The reaction took place when GSH-pDA-IONPs were successively added in NaNO2 solution at 37 �C and pH 2.5 for 30 min in the dark. UV-vis spectra were recorded to see the formation of S-nitrosation and the resultant nanoparticles were finally washed with PBS buffer and re-dispersed in PBS.

IDA magnetic Beads

https://onlinelibrary.wiley.com/doi/pdf/10.1002/bmc.3584

Synthesis of Fe3O4 particles  (solvothermal method.)

The Fe3O4 particles were prepared via a solvothermal method as described previously (Xu et al., 2006). Briefly, 5.4 g of FeCl3 · 6H2O was first dissolved in 200mL of ethylene glycol under violent stirring. A clear yellow solution was obtained after filtration. Then 4.0 g of PEG 10000 and 14.4 g sodium acetate were added to this solution. The mixture was stirred vigorously for 30 min and then sealed in a Teflon-lined stainless-steel autoclave (200mL capacity). The autoclave was heated to and maintained at 200 °C for 12 h, and then allowed to cool to room temperature. The black products were washed several times with ethanol and deionized water and dried at 60 °C for 8 h.

Synthesis of Fe3O4@SiO2 (sol–gel method)

The core–shell Fe3O4@SiO2 nanocomposites were prepared using a modified method from Stöber et al. (1986). The core–shell Fe3O4@SiO2 magnetic microspheres were synthesized by a sol–gel process to coat a thin layer of dense amorphous silica on Fe3O4 nanoparticles. Briefly, an ethanol dispersion of the magnetite particles (150mg) was added to a beaker and washed three times with deionized water with help of the permanent magnet. Then, 50mL of 0.1mol/L HCl solution was added followed by ultrasonication for 30min. The supernatant was decanted and the superparamagnetic Fe3O4 nanoparticles were added to a three-neck round-bottom flask and dispersed in 80mL ethanol and 20mL deionized water by ultrasonication for about 10min. Under continuous mechanical stirring, 1.0mL of ammonium hydroxide (28 wt%) and 0.8mL of TEOS were consecutively added to the reaction mixture. The reaction was allowed to proceed at 30 °C for 6 h under continuous stirring. The resultant product was obtained by magnetic separation with help of the permanent magnet and was thoroughly washed with ethanol and deionized water six times (50mL each time). The product was then dried under vacuum at 60 °C for 8 h.

silica was coated on the Fe3O4 using a sol–gel method to form Fe3O4@SiO2 core–shell microspheres through hydrolysis of TEOS according to the Stöber method. Because the surface of Fe3O4 has a strong affinity toward silica, no primer was required to promote the deposition and adhesion of silica. The growth of silica shells on Fe3O4 nanoparticles involved the base-catalyzed hydrolysis of TEOS and subsequent condensation of silica onto the surfaces of Fe3O4 cores. Several parameters (such as the growth time and the concentration of ammonia catalyst or water) can be employed to control the thickness of silica shell on the Fe3O4 surface. It is found that the particle size can be conveniently adjusted by changing the concentration of TEOS precursor.

Preparation of IDA-modified Fe3O4@SiO2 magnetic microspheres

The IDA-modified Fe3O4@SiO2 magnetic microspheres were prepared using the Fe3O4@SiO2 magnetic microspheres modified with IDA as chelating ligand and are shown in Scheme 1. Briefly, 2.5 g IDA was dissolved in 50mL of a 2mol/L solution of Na2CO3, and the pH was adjusted to 10.0 with 10mol/L NaOH. The IDA solution was stirred for several minutes in an ice bath and then3-glycidoxypropyltrimethoxysilane (1.6mL) was added into the solution slowly. After 30min of stirring at 0°C, the reaction mixture was allowed to warm up to 65°C with stirring. After 6 h, the reaction solution was cooled to room temperature and the pH was adjusted to 6.0 with concentrated HCl. One gram of Fe3O4@SiO2 magnetic microspheres was added to the above solution and ultrasonicated for 10min. The suspension was heated at 95°C for 2 h with stirring. The resulting nanoparticles denoted superparamagnetic IDA silica were separated with help of the permanent magnet and washed thoroughly with deionized water six times (50mL each time). Charging of Fe3O4@SiO2–IDA magnetic microspheres with metal ions Charging of Fe3O4@SiO2–IDA magnetic microspheres with metal ions (eg. Cu2+, Ni2+ and Zn2+ ions) was performed as follows: 60mg of Fe3O4@SiO2–IDA magnetic microspheres were mixed with 50mL of aqueous solution of CuSO4 (10mg/mL). The mixture was stirred magnetically at 100 rpm for 3 h. Then the excess unbound Cu2+ was removed with water. The same method was followed to chelate Ni2+ or Zn2+ using the aqueous solution of NiSO4 or ZnSO4 (10mg/mL).

Charging

60mg of Fe3O4@SiO2–IDA magnetic microspheres were mixed with 50mL of aqueous solution of CuSO4 (10mg/mL). The mixture was stirred magnetically at 100 rpm for 3 h. Then the excess unbound Cu2+ was removed with water. The same method was followed to chelate Ni2+ or Zn2+ using the aqueous solution of NiSO4 or ZnSO4 (10mg/mL).

Regeneration of Fe3O4@SiO2@IDA–metal ions magnetic microspheres: 50 mM EDTA,

Highly selective magnetic affinity purification of histidine-tagged proteins by Ni2+ carrying monodisperse composite microspheres

Synthesis of magnetic SiO2 (SiO2@Mag-SiO2) microspheres The poly(glycidyl methacrylate), poly(GMA), seed latex 2.1 mm in size was synthesized by dispersion polymerization of GMA.32 Monodisperse-porous poly(methacrylic acid-co-ethylene dimethacrylate), poly(MAA-co-EDMA) microspheres 6 mm in size were obtained by using multi-step microsuspension polymerization technique.33,34 The magnetization of poly(MAA-co-EDMA) microspheres was performed as described elsewhere.35 The magnetic poly(MAA-co-EDMA) microspheres were used as a template for synthesis of monodisperse, magnetic SiO2 microspheres. Briey, the poly(MAA-co-EDMA) microspheres were dispersed in an aqueous solution containing TBAI, ammonia, 2-propanol and distilled water for 1 hour under mechanical stirring. TEOS solution (in IPA containing 50 w/w%) was added dropwise into the solution and stirred for 24 h at R.T. The formed silica-gel/poly(MAA-co-EDMA) composite microspheres were then separated by a magnet and washed with IPA and distilled water 3 times. The magnetic microspheres were dried in vacuum at 60 C for 24 h. The calcination was carried out at 450 C for 10 h for formation of the magnetic, monodisperse SiO2 microspheres. Finally, SiO2 coating onto the MagSiO2 microspheres (SiO2@Mag-SiO2) was done with the same process as described above.31 2.3 Synthesis of iminodiacetic acid attached magnetic SiO2 (IDA–GLYMO@SiO2@Mag-SiO2) microspheres The synthesis of iminodiacetic acid–(3-glycidoxypropyl)trimethoxysilane (IDA–GLYMO) precursor was performed by using a modied form of the protocol reported previously:36 IDA (0.53 g) was dissolved in 10 mL of DDI water, and pH of solution was adjusted to 11.0 with 10.0 M NaOH. The obtained solution was transferred into a Pyrex® reactor placed in an ice-bath at 0 C, and 0.4 mL of GLYMO was slowly added under stirring. The solution was heated to 65 C for 6 h with stirring, subsequently placed into an ice-bath for 10 min to decrease the temperature to 0 C. 0.4 mL of GLYMO was again added and the temperature was increased to 65 C for another 6 h under stirring. Finally, pH of IDA–GLYMO solution was adjusted to 3 with concentrated HCl. In order to attach IDA–GLYMO precursor onto the SiO2@Mag-SiO2 microspheres, 0.2 g of microspheres were added to IDA–GLYMO solution at pH 3 and the resulting dispersion was heated at 95 C for 2 h with mechanical stirring. The resulting IDA–GLYMO@SiO2@Mag-SiO2 microspheres were extensively washed with DDI water and nally dispersed in DDI water. 2.4 Ni2+ attachment onto IDA–GLYMO@SiO2-Mag-SiO2 microspheres Ni2+-immobilized IDA–GLYMO@SiO2@Mag-SiO2 (Ni2+–IDA– GLYMO@SiO2@Mag-SiO2) microspheres were prepared by the incubation of IDA–GLYMO@SiO2@Mag-SiO2 microspheres (20 mg) in 0.25 M aqueous NiCl2 solution at RT for 2 h under gentle stirring. The obtained Ni2+–IDA–GLYMO@SiO2@Mag-SiO2 microspheres were collected by magnetic separation and washed with distilled water several times to remove free Ni2+ ions. In this case, each Ni2+ ion coordinates to more than one carboxyl groups (–COOH) via metal(II) carboxyl chemistry.

Magnetic core nanoparticles synthesis

https://bomb.bio/

A simple co-precipitation protocol for preparation of magnetic nanoparticles (MNP). It relies on forming Fe3O4 magnetic particles during co-precipitation of FeCl2 and FeCl3 in alkali solutions [1]. The formed MNPs have a diameter of ~5-20 nm. Oxygen is known to interfere with the synthesis, therefore the reactions can be either flushed with N2 for at least 30 minutes, performed immersed in argon or degassed and preheated to 80 °C right before the synthesis. 

Iron (II) chloride 4-hydrate puriss ≥99% (FeCl2 · 4 H2O) Honeywell/Fluka 44939

Iron (III) chloride, anhydrous, pure (FeCl3) Roth Chemicals 5192.3

Sodium hydroxide, ≥99% (NaOH) Roth Chemicals 6771.1

37% HCl fuming Roth Chemicals 4625.1

Ammonia solution (NH4OH, 25%) EMD Millipore 1.05432

Buffers and solutions

2 M NaOH (prepare 0.5 L) –can be stored at RT for at least 12 months

0.1 M HCl (prepare 0.5 L) – can be stored at RT for at least 12 months

Fe2/3 solution (prepare freshly 60 ml)

0.333 M FeCl3 (3.24 g FeCl3)

0.167 M FeCl2 (2 g FeCl2 · 4 H2O)

Dissolve with magnetic stirring in 60 ml of 0.1 M HCl solution

Equipment

Fume hood Magnetic stirrer with heating (e.g. IKAMAG REO)

Strong neodymium permanent magnet (e.g. Neodymium disc magnet 80x10 mm grade N45 adhesive force ~300 kg – be extremely careful when using such strong magnets!)

Sterile plastic 0.5 L bottle (e.g. Corning® Costar®, Nunc® T175 uncoated PS flask)

Protective clothing (lab coat, eye protection, hand gloves)

Experimental setup

All procedures can be performed under inert argon atmosphere (alternatively purging the system with high purity N2 for 30 minutes should be sufficient. We generally perform the reaction at atmospheric oxygen levels yet degas and heat up the solutions before use)

1 Assemble the reaction bottle and the dripping funnel and place on a magnetic stirrer, add 100 ml of degassed 2 M NaOH to the collection flask on the bottom and the Fe2/3 solution to the dripping funnel. Heat up the flask containing the 2 M NaOH solution to 80-85 °C. 10 min

2 Add drop wise the 60 ml Fe2/3 solution into 100 ml of preheated (80-85 °C) 2 M NaOH solution under vigorous stirring (>400 rpm) 20 min

Black precipitate of Fe3O4 (FeO·Fe2O3) is formed

3 After the complete Fe2/3 solution was added, add 10 ml of 25% ammoniac to the reaction mixture and stir for a further 30 minutes and then cool to RT 30 min

4 Transfer the solution to a sterile plastic container and magnetically pellet the black precipitate (MNPs), discard the supernatant 5 min

5 Resuspend in 200 ml of ddH2O, pellet magnetically and discard supernatant. Repeat for a total of 5 washes (or until the pH of the wash reaches ~7) 1 h

6 After the last wash resuspend the MNPs in 30-40 ml of ddH2O and transfer to a fresh 50 ml tube

End To measure the synthesis yield, magnetically pellet the MNPs, remove the water and weigh the product wet-mass, alternatively flash freeze the pelleted MNPs in liquid nitrogen and lyophilise and measure the dry mass ~2 h (~1 h hands-on) Lyophilised MNPs under N2 or argon atmosphere can be stored in a closed container at RT for a longer time, the wet MNPs slowly oxidise over time, therefore we functionalise them further within a week

Modifications Different protocols provide MNPs with different properties. This protocol was selected and optimized for robustness and because it yields relatively small sized magnetic particles (~5-20 nm in diameter as judged by the TEM images). However, a plethora of other methods can be employed for the synthesis of suitable MNPs as reviewed in [2].

No precipitate formed

Use freshly made alkali solutions, correct iron salts at indicated concentrations; Preheat the alkali solution to at least 70 °C or degas before adding the Fe2/3 solution

Precipitate not magnetic

Make sure you are using freshly made Fe2/3 solution and that you have used correct iron salts for the mixture

Coating ferrite MNPs with silica oxide

https://bomb.bio/

The silica-coated magnetic beads are synthesised by a modified protocol including the hydrolysis of tetraethyl orthosilicate (TEOS) on the surface of ferrite magnetic core particles according to Stöber et al. 1968 [1]

Chemicals
Ethanol (C2H6O, 99.9 %) Riedel-de Haёn 34963
Tetraethyl orthosilicate (≥99%) (GC) Aldrich 86578 
Ammonia solution (NH4OH, 25%) EMD Millipore 1.05432

Equipment and setup
Fume hood
Heated magnetic stirrer (e.g. IKAMAG REO)
Strong neodymium permanent magnet (e.g. NdFeB N45 40x40x20 mm)
Sterile plastic bottles 

All procedures can be performed under inert N2 atmosphere or standard conditions
1 Mix 45 ml of TEOS in 2 L ethanol in a large bottle (2.5 litre) using a magnetic stirrer and mix for 15 minutes at 300-400 rpm 15 min 
2 Add 22.5 g (wet mass ~1.2 g dry) of magnetic core particles prewashed with ethanol and stir for 15-30 min 30 min
3 Add 400 ml of pure water and 50 ml of 25% ammonia solution and stir for another 30 min 30 min 
4 Switch on the heating and allow the solution to heat up to ~80 °C with constant stirring, allow the reaction to proceed for >4 h (ideally o/n) 4 hours to o/n 
5 Cool down the solution 15 min 
6 Separate the coated MNPs using a strong neodymium magnet 10 min 
7 Wash twice with pure water 15 min 
8 Wash twice with pure ethanol 15 min 
9 Wash with pure water until the pH of the solution becomes neutral (3-4 times) 30 min 
Check the yield by weighing the wet mass of the beads ~12 h (2 h hands-on)

Modifications

By using different ratios of core particles and TEOS one can control the thickness of the glass layer and consequently the size of the particles. The standard ratio used in the above protocol is: 1 g of magnetic core particles, 2 ml TEOS, 20 ml of ethanol, 0.5 ml of 25 % ammonia solution, 4 ml of water, which results in particles with an average size of ~400 nm. Increasing or decreasing the TEOS to beads ratio results in formation of other sizes of coated particles

Troubleshooting

Problem 
Brown colour of the reaction, low yield of beads retained
Solution 
The uncoated magnetic core particles get slowly oxidized during storage, prepare fresh core particles and redo the reaction

Problem 
Large amounts of white precipitate
Solution

TEOS hydrolyses spontaneously in water solution, thus forming silica nanoparticles. Use fresh TEOS for the reaction

Coating ferrite MNPs with carboxyl groups (methacrylic acid)

https://bomb.bio/

Coating ferrite magnetic nanoparticles (MNPs, BOMB protocol #1.1) with carboxyl groups. The carboxyl-coated magnetic nanoparticles are synthesized by the classical radical polymerisation of methyacrylic acid (MAA) on the surface of ferrite magnetic core particles (protocol modified from Yu et al. [1]). 

Chemicals

Ethanol (C2H6O, 99.9 %) Honeywell/ Riedel-de Haёn 34963

Methacrylic acid, MAA (≥99%) (GC) Aldrich 155721

https://www.tcichemicals.com/eshop/en/us/commodity/M0079/

500g $26

Potassium persulfate (K2S2O8, ≥99%) Sigma-Aldrich 216224

Sodium dodecyl sulfate / SDS (C12H25NaO4S) Roth Chemicals CN30.3 2

Equipment and setup

Fume hood

Heated magnetic stirrer (e.g. IKAMAG REO)

Strong neodymium permanent magnet (e.g. NdFeB N45 40x40x20 mm)

Sterile plastic bottles

All procedures can be performed under inert N2 atmosphere or atmospheric oxygen conditions

1 Add 1 g (wet mass) of the synthesized iron oxide MNPs dispersed in 45 ml of water and 1.15 g sodium dodecyl sulfate to 200 ml of purified, degassed water in a 250 ml flask with stirring 5 min

2 Heat up the reaction solution to 70 °C 30 min

3 Add 0.96 ml of MAA into the flask. The pH value decreases to about 3, 5 min

4 Equilibrate the reaction mixture for about 45 min while keeping the temperature 45 min 

5 Add 1.98 g of initiator K2S2O8 to the solution 15 min

6 Let the polymerization reaction progress at 70 °C for 2 h 2 hours

7 Separate the coated MNPs using a strong neodymium magnet 15 min

8 Afterwards cool the reaction to room temperature 15 min

9 Remove free MAA and PMAA (poly(methacrylic acid) from the coated magnetic particles by a magnetic particle concentrator at room temperature 30 min

10 Disperse the isolated magnetic nanoparticles in deionized water in an ultrasonic bath, followed by magnetic extraction 5 min

11 Wash the beads with ddH2O at least 5 times or until the detergent is completely removed 1 h

12 Dispersed the magnetic particles in 250 ml of deionized water 5 min End Check the yield by weighing the wet mass of the beads ~7 h (2 h handson)

Store @ RT for up to 1 year

A 45 ml portion of the synthesized iron oxide colloid solution and 1.15 g sodium dodecyl sulfate were added to 200 ml of purified water in a 250 ml flask with mechanical stirring. After the reaction solution was heated to 70 oC, 0.96 ml of MAA was introduced into the flask. The pH value of the system was about 3. The reaction mixture was equilibrated for about 45 min at the same temperature before 1.98 g of initiator K2S2O8 was added to the solution. The polymerization was carried out at 70 oC in argon for 2 h, followed by cooling the reaction system to room temperature. To remove free MAA and PMAA, magnetic particles in the colloid solution obtained were extracted by a magnetic particle concentrator (Dynal MPC1-50, Dynal Biotech, France) at room temperature. The isolated magnetic nanoparticles were dispersed in deionized water under ultrasonication, followed by magnetic extraction. The washing process was repeated at least 5 times. A portion of the washed magnetic particles was freeze-dried for subsequent characterization.

Fig 1: Carboxyl-coated MNPs appear brownish (left vial) when compared to silica-coated MNPs (right vial, BOMB protocol #2.1)

References

1. Yu S, Chow CM. Carboxyl group (-CO2H) functionalized ferrimagnetic iron oxide nanoparticles for potential bio-applications. J Mater Chem. Royal Society of Chemistry; 2004;14: 2781–2786. doi:10.1039/b404964k

Synthesis of NTA-PEG3400-Si(OMe)3 NTA-PEG3400-VS

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533260/

NHS-PEG3400-VS (0.85 g, 0.25 mol) and NTA (0.33 g, 1.25 mmol) were dissolved in 8.5 ml of DMSO. Triethylamine (TEA) (2.00 ml, 14.30 mmol) was added and the solution stirred at room temperature for 24 h before quenching with 2N HCl (28.3 mmol). The solution was then dialyzed for 24 h against 1 L of distilled water using a dialysis membrane (Spectra/Por, MWCO 1000) and the retentate lyophilized. The NTA-PEG3400-VS product was stored at −20 °C until use.

NTA-PEG3400-Si(OMe)3 Silanation of NTA-PEG3400-VS was performed by free radical addition of mercaptopropyltrimethoxysilane (MPTS) to the vinyl sulfone substituent as shown in Scheme 1.34,35 NTA-PEG3400-VS and an eight-fold excess of freshly distilled MPTS were added to toluene in a round bottom flask, followed by the addition of a 0.4-fold excess of 2,2′-azobisisobutyronitrile (AIBN) relative to NTA-PEG3400-VS. The mixture was stirred for 24 h at 60 °C under a N2 atmosphere before precipitation of the product with anhydrous Et2O. The precipitate was filtered, washed with excess Et2O, and dried under reduced pressure to give NTA-PEG3400-Si(OMe)3 as a white powder in 53% yield. 1H NMR (CDCl3): δ 3.9-3.15 (br, CH2CH2O), 2.45 (t, J=6.0 Hz, NHCH2), 1.68-1.14 (br, NHCH2(CH2)3CHCOOH), and 0.6 (CH2Si).

Surface preparation and characterization Silicon wafers or cover glasses were immersed into fresh piranha solution for 1 h and rinsed by bath sonication in deionized water. Piranha solution was prepared by mixing pure sulphuric acid and hydrogen peroxide (30% solution) in a 7:3 volume ratio. Piranha solutions are potentially explosive and highly corrosive! Handle with extreme caution! The clean substrates were dried under N2 gas and then immersed in toluene solutions of mPEG5000-Si(OMe)3 or NTA-PEG3400-Si(OMe)3 (10 mg PEG derivative/ml) for 16 h at 60 °C.35 The NTA-PEG3400-VS-modified surfaces were prepared as follows: piranha cleaned surfaces were immersed in 1% MPTS in toluene for 4 h and washed with toluene. The dried MPTS-modified surface was then incubated in NTA-PEG-VS (10 mg/ml) in 0.01M phosphate buffered saline (PBS) for 16 h at room temperature.36

iron oxide silica particles

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854921/

iron oxide silica particles To synthesize the Fe3O4@Silica for RNA extraction, the following chemicals of analytical reagent grade were received and used without further purification. Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), styrene, glycidyl methacrylate (GMA), divinylbenzene (DVB), azobisisobutyronitrile (AIBN), polyacrylic acid, guanidine hydrochloride (>99%) and ethanol anhydrous were obtained from Sigma Aldrich. Ammonia in water 28–30% w/w, sodium citrate tribasic dihydrate, nitric acid (90%) were obtained from BDH. Tetraethyl Orthosilicate (TEOS), 3-glycidyloxypropyltrimethoxysilane (GTMOS), (3-Aminopropyl)triethoxysilane (APTES) were purchased from TCI (USA). All aqueous solutions were prepared with deionized water obtained from a Milli-Q Integral system (resistivity = 18 MΩ cm, EMD Millipore). Zymo MagBinding® Beads was obtained from Zymo Research, USA for comparison in RNA binding. Dynabeads® MyOneTM, M280 from Thermofisher were used as the microscopic reference sizing materials, employed at 800x magnification.

2.2. Synthesis of Fe3O4@Silica from iron oxide nanoparticles (Fe3O4) as seeds Fe3O4 nanoparticles were prepared by the basic co-precipitation method. 4.46 g (16.6 mmol) FeCl3·6H2O and 1.6 g (8.1 mmol) of FeCl2·4H2O were dissolved in 80 ml of deionized water at room temperature. The solution was degassed by bubbling with Ar for 5 min and magnetic stirring in conical flask. 10 ml of ammonia 28–30% w/w was added dropwise to the solution and stirred for another 10 min. at room temperature. Following with heating for 1 h at 90 °C, iron oxide magnetite (Fe3O4) formed was cooled down to room temperature and magnetically separated, washed with deionized water until pH 8 reached.

Fe3O4 particles formed in above method were treated with 2 M nitric acid for 5 min with stirrer mixing at room temperature, we believe this treatment can stabilize the Fe3O4 particle surface [25, 26]. After the reaction, the particles were washed with deionized water and magnetically separated until the pH reached ∼2.5. Then, 10 ml aqueous solution of 0.5 M sodium citrate was added to the 30 ml particles suspension. The resulting solution was stirred for 1 h at room temperature to prepare the surface for silica coating. Finally, the citrate surface stabilized particles were magnetically recovered, washed thoroughly with deionized water and freeze-dried. Dried beads weighed 1.77 g (MW 231.53, 7.6 mmol); yield = 88%. Dynamic light scattering (DLS) and zeta potential measurements were obtained using a Malvern ZETASIZER NANO ZSP. The sample concentrations were adjusted to ∼0.1% w/v particles in deionized water. The samples were sonicated in ultrasonic bath for 1 min and pre-equilibrated for 3 min at 25 °C prior to each measurement. Zeta potential measured was −28.5 mV. Scanning Electron Microscopy (SEM) analysis was obtained from FEI Helios Nanolab 660. SPION stabilization with citrate is a protocol inspired by Khosroshahi et al and Digigow et al [25, 26]. This 1-pot, 2-step procedure leads to citrate surface-stabilized Fe3O4 nanoparticles that can be stored in dry form at 4 °C.

To prepare the silica glass surface of the above citrate- Fe3O4 particles. Re-suspension and sonication of these dry particles in 10% v/v water in ethanol, followed by silica shell formation with hydrolysis of tetraethoxysilane (TEOS) under alkaline condition using 28–30% ammonia, results in silica magnetic particles that can be magnetically separated without further purification (Fig. 1). At the end, the silica coated magnetic microparticles were rinsed with ethanol and dried in vacuum. Dried microparticles weighed 3.4 g.

Different silica mass ratio were obtained by using different reactants ratio of TEOS:NP, ranging from 2:1, 4:1 to 6:1. Corresponding mass ratios of silica coating ranged from 45%, 55% and 62% were obtained. A mass ratio of TEOS vs starting particles NP of 1:1 was not pursued because of the possibility of incomplete silica enclosure on the Fe3O4 magnetic particles. And we observed that a higher silica mass ratio than 55% was not preferred as the additional weight of the silica shell resulted in a slower magnetic response of the coated Fe3O4@Silica particles.

2.3. Formulation of Fe3O4@Silica for RNA extraction in Zika assays Preparation of 10% w/v of the Fe3O4@Silica in 1 M Gu.Cl buffer for Zika assays was done by suspension of 100 mg of the Fe3O4@Silica prepared above in 500 μl deionized water, then added with 2 M Gu.Cl buffer to make up to 1.0 ml final volume. Zeta potential of this formulated Fe3O4@Silica was measured as −4.84 mV.

2.4. Alternative methods for synthesis of Fe3O4@Silica from polymer seeds Alternative methods to prepare Fe3O4@Silica beads were also tested; for example, polymerizing a mixture of (1) TEOS with γ-aminopropyl-triethoxysilane (APTES); (2) TEOS with (3-mercaptopropyl)trimethoxysilane (MPTMS); and (3) amino surface of magnetic polystyrene particles reacted with (3-glycidyloxypropyl)trimethoxy silane (GLYMO). In typical examples, in-house synthesized amine surface polymer microparticles with approximate 2 μm was used as seeds. 0.5 g of seeds were reacted with 0.2 ml 3-glycidyloxypropyltrimethoxysilane (GTMOS) for 1 h at room temperature in 5 ml ethanol, followed with 0.1 ml tetraethyl orthosilicate (TEOS) or a mixture of 0.1 ml tetraethyl orthosilicate (TEOS) and 0.2 ml (3-Aminopropyl)triethoxysilane (APTES) in 0.2 ml ammonia 28–30% w/w for 4 h at 90 °C (Fig. 3). After reaction, the magnetic particles were magnetically separated and washed with ethanol (5 times) with centrifugation at 3000 × g or separated magnetically. Zeta potential measured microparticles with silica surface formed from (TEOS:APTES, 2:1, v/v) = +39.2 mV microparticles with silica surface formed from TEOS only = +15.3 mV. This preliminary study on the ratio of TEOS:APTES relationship with surface Zeta potential demonstrates the fine tunable controls on the surface charges by the design of combination of silylating reagents used.

Fe3O4/PMG/IDA−Ni2+ Nanoparticles

https://www.nature.com/articles/srep41741

γ-Methacryloxypropyltrimethoxy-silane (MPS)

Glycidyl methacrylate (GMA)

2, 2′-Azobis (2-methylpropionitrile) (AIBN)

The Fe3O4 particles with a core size around 200 nm were synthesized through a modified solvothermal reaction. Typically, FeCl3·6H2O (1.350 g), NH4Ac (3.854 g) and trisodium citrate dihydrate (0.400 g) were dissolved in ethylene glycol (70 mL). The mixture was stirred vigorously for 1 h at 160 °C to form a homogeneous black solution, and then transferred into a Teflon-lined stainless-steel autoclave (100 mL capacity). The autoclave was heated at 200 °C and maintained for 16 h, and then it was cooled to room temperature. The black product was washed with ethanol and separated from the solvent by using a magnet. The cycle of washing and magnetic separation was repeated for several times. The final product was dispersed in ethanol for further use.

Modification of the Fe3O4 with MPS
The modification of Fe3O4 with MPS was achieved by adding 40 mL of ethanol, 10 mL of deionized water, 1.5 mL of NH3·H2O, and 0.6 g of MPS into the Fe3O4 particles ethanol suspension and vigorously stirring the mixture for 24 h at 70 °C. The obtained product was separated by using a magnet and washed with ethanol to remove excess MPS. The resultant Fe3O4-MPS nanoparticles were dried in a vacuum oven at 40 °C until they reached a constant weight.

Synthesis of Fe3O4/PMG Core/Shell Nanoparticles
Coating a PMG layer onto Fe3O4-MPS nanoparticles was executed by the distillation-precipitation polymerization (DPP) of GMA, with MBA as the cross-linker and AIBN as the initiator, in acetonitrile. Typically, Fe3O4-MPS seed nanoparticles (50 mg) were dispersed in acetonitrile (40 mL) in a dried single-necked flask (100 mL capacity) under ultrasonic condition for 3 min. Then a mixture of GMA (150 mg), MBA (150 mg), and AIBN (6 mg) was added to the flask to initiate the polymerization. The flask, submerged in a heating oil bath, was attached to a fractionating column, Liebig condenser, and a receiver. The reaction mixture was heated from room temperature to the boiling state within 30 min and the reaction was ended after acetonitrile (20 mL) was distilled from the reaction mixture within 1 h. The obtained Fe3O4/PMG nanoparticles were collected by magnetic separation and washed with ethanol and water repeatedly.

Preparation of Fe3O4/PMG/IDA-Ni2+ Nanoparticles
IDA (0.33 g) and NaOH (0.2 g) were dissolved in 20 mL of deionized water under stirring. 2 M NaOH solutions were used to adjust the pH of the solution. Then Fe3O4/PMG nanoparticles (50 mg) were added to the solution, and the mixture was stirred vigorously for 12 h at 80 °C. The obtained Fe3O4/PMG/IDA nanoparticles were separated by a magnet and washed by ethanol and water for several times. Subsequently, 50 mg of Fe3O4/PMG/IDA nanoparticles was added to a 10 mL of 0.1 M NiCl2 solution and stirred for 2 h at room temperature. The product was separated by a magnet from the solution and washed several times with water. The resultant Fe3O4/PMG/IDA-Ni2+ nanoparticles were dried in a vacuum oven at 40 °C.

Excellgen Protocol

Reagents

(3-Glycidyloxypropyl)trimethoxysilane: 

https://www.tcichemicals.com/eshop/en/us/commodity/G0210/

(3-acryloxypropyl) trimethoxysilane, 100g $38

Sigma https://www.sigmaaldrich.com/catalog/product/aldrich/440167  100 ML 47.30

(3-Glycidyloxypropyl)trimethoxysilane (GPTMS) is a bifunctional organosilane with three methoxy groups on one side and an epoxy ring on the other. The methoxy groups bind well with glass substrates creating a 3D matrix. The epoxy group is reactive with amides, alcohols, thiols and acids. GPTMS is highly reactive in water and can be used as a linking agent between the surface of the silica and the polymeric matrix.

 IDA, https://www.tcichemicals.com/eshop/en/us/commodity/I0006/ I0006   Iminodiacetic Acid, 500g $41

(5S)-N-(5-Amino-1-carboxypentyl)iminodiacetic Acid, 113231-05-3, for making NTA

https://www.chemsrc.com/SupplierProduct/113231-05-3_453224.html

https://www.trc-canada.com/product-detail/?CatNum=A603250, 10 g

http://www.9dingchem.com/113231-05-3.html

Preparation of IDA-modified Fe3O4@SiO2 magnetic microspheres
Dissolve   2.5 g IDA in 50mL of a 2mol/L solution of Na2CO3, adjust pH   to 10.0 with 10mol/L NaOH. 

Stir the IDA solution for several minutes in an ice bath 
Add 1.6mL of 3-glycidoxypropyltrimethoxysilane   into the solution slowly. 
After 30 min of stirring at 0°C, 
Warm up to 65°C while  stirring. 
After 6 h, cool the reaction solution  to room temperature

Adjust pH to 6.0 with concentrated HCl. 

Add 1g of Fe3O4@SiO2 magnetic microspheres   to the above solution 

Sonicat for 10min. 

Heat the suspension  at 95°C for 2 h with stirring. T

Separate beads with  a permanent magnet
Wash thoroughly with deionized water six times (50mL each time).
Charge of Fe3O4@SiO2–IDA magnetic microspheres with metal ions: Mix 60mg of Fe3O4@SiO2–IDA magnetic microspheres   with 50mL of aqueous solution of CuSO4 or NiSO4 or ZnSO4 (10mg/mL). 
Stir the mixture agnetically at 100 rpm for 3 h.