Background Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties.
Background
Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties.Methods Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy.
Methods
Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy.Results The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles
Results
The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94–100% while thiophene acetylation was 66–72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticlesConclusion Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications
Conclusion
Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications

Background The immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials or devices [ 1 ]. Enzyme immobilization for example, is a desired biological procedure because of the possible application of immobilized enzymes in continuous operations, product purification, and catalyst recycling [ 2 ]. Furthermore, immobilization provides many advantages such as enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth [ 3 ]. In the last decade, nanosize materials have been widely used as support for this purpose. Among these materials, magnetic nanoparticles are very popular when used in conjunction with biological materials including proteins, peptides, enzymes [ 4 - 9 ] antibodies and nucleic acids [ 8 ], because of their unique properties [ 4 - 9 ]. The ability to tract magnetically labeled entities or target organelles using magnetic force offer the opportunity to conduct biological operations with increased specificity. Magnetite (Fe 3 O 4 ) are biocompatible superparamagnetic materials that have low toxicity and strong magnetic properties [ 5 ]. They have been widely used for  in  vivo examination including magnetic resonance imaging, contrast enhancement, tissue specific release of therapeutic agents, hyperthermia [ 10 , 11 ], magnetic field assisted radionucleide therapy [ 11 ], as well as  in  vitro binding of proteins and enzymes [ 4 - 8 ]. Magnetite nanoparticles have been used as support material for binding of enzymes including yeast alcohol dehydrogenase [ 4 ] and lipase [ 5 ] directly via carbodiimide activation. This method brought about considerable promise because of its simplicity and efficiency. Recently, γ-Fe 2 O 3 magnetic nanoparticles were used for binding  Candida rugosa  lipase after acetylation of thiophene functionalized nanoparticles, or through nitroso-derivative formed on the surface of the particles by reacting nitroso tetrafluoroborate in methylene chloride. Both methods and more effectively lipase immobilized on acetylated nanoparticles exhibited long term stability. Glucose oxidase (GOX, β-D-glucose oxygen 1-oxidoreductase, EC 1.1.3.4) is a homodimer flavoprotein containing two active sites per molecule [ 12 , 13 ]. It catalyses the oxidation of β-D-glucose to gluconic acid, concomitant with the reduction of oxygen to hydrogen peroxide. Glucose oxidase has been used to test various types of enzyme immobilization, and is the most commonly studied in the construction of biosensors for glucose assay development [ 12 , 14 , 15 ]. A more recent study [ 16 ] examined the activity of cholesterol oxidase activity using carbodiimide activation. Here, we report the stability and enzymatic activity of glucose oxidase immobilized onto Fe 3 O 4  magnetic nanoparticles using two binding methods, the direct binding via carbodiimide activation of amino functionalized particles and binding to thiophene-functionalized acetylated nanoparticles. A comparison of the stability and activity of glucose oxidase immobilized to magnetite using different protocols will lay the foundation for magnetic immunoassays. The size and structure of the nanoparticles were characterized using Transmission electron microscopy (TEM) and Fourier Transform Infrared (FTIR) spectroscopy, respectively. The stability, activity, and kinetic behavior of bound glucose oxidase were also examined.
Background
The immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials or devices [ 1 ]. Enzyme immobilization for example, is a desired biological procedure because of the possible application of immobilized enzymes in continuous operations, product purification, and catalyst recycling [ 2 ]. Furthermore, immobilization provides many advantages such as enhanced stability, easy separation from reaction mixture, possible modulation of the catalytic properties, and easier prevention of microbial growth [ 3 ].In the last decade, nanosize materials have been widely used as support for this purpose. Among these materials, magnetic nanoparticles are very popular when used in conjunction with biological materials including proteins, peptides, enzymes [ 4 - 9 ] antibodies and nucleic acids [ 8 ], because of their unique properties [ 4 - 9 ]. The ability to tract magnetically labeled entities or target organelles using magnetic force offer the opportunity to conduct biological operations with increased specificity.Magnetite (Fe 3 O 4 ) are biocompatible superparamagnetic materials that have low toxicity and strong magnetic properties [ 5 ]. They have been widely used for  in  vivo examination including magnetic resonance imaging, contrast enhancement, tissue specific release of therapeutic agents, hyperthermia [ 10 , 11 ], magnetic field assisted radionucleide therapy [ 11 ], as well as  in  vitro binding of proteins and enzymes [ 4 - 8 ].ininMagnetite nanoparticles have been used as support material for binding of enzymes including yeast alcohol dehydrogenase [ 4 ] and lipase [ 5 ] directly via carbodiimide activation. This method brought about considerable promise because of its simplicity and efficiency. Recently, γ-Fe 2 O 3 magnetic nanoparticles were used for binding  Candida rugosa  lipase after acetylation of thiophene functionalized nanoparticles, or through nitroso-derivative formed on the surface of the particles by reacting nitroso tetrafluoroborate in methylene chloride. Both methods and more effectively lipase immobilized on acetylated nanoparticles exhibited long term stability. Glucose oxidase (GOX, β-D-glucose oxygen 1-oxidoreductase, EC 1.1.3.4) is a homodimer flavoprotein containing two active sites per molecule [ 12 , 13 ]. It catalyses the oxidation of β-D-glucose to gluconic acid, concomitant with the reduction of oxygen to hydrogen peroxide. Glucose oxidase has been used to test various types of enzyme immobilization, and is the most commonly studied in the construction of biosensors for glucose assay development [ 12 , 14 , 15 ]. A more recent study [ 16 ] examined the activity of cholesterol oxidase activity using carbodiimide activation.Candida rugosaHere, we report the stability and enzymatic activity of glucose oxidase immobilized onto Fe 3 O 4  magnetic nanoparticles using two binding methods, the direct binding via carbodiimide activation of amino functionalized particles and binding to thiophene-functionalized acetylated nanoparticles. A comparison of the stability and activity of glucose oxidase immobilized to magnetite using different protocols will lay the foundation for magnetic immunoassays. The size and structure of the nanoparticles were characterized using Transmission electron microscopy (TEM) and Fourier Transform Infrared (FTIR) spectroscopy, respectively. The stability, activity, and kinetic behavior of bound glucose oxidase were also examined.Materials and methods Glucose oxidase (specific activity 200 units/mg protein) from  Aspergillus  niger was purchased from VWR international (Pittsburgh, USA). Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide, sodium hydroxide, acetic anhydride, glucose, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 % and iron (III) chloride hexahydrate 99%, were obtained from Sigma-Aldrich St Louis (USA). 11-bromoundecanoic acid was obtained from TCI America Portland, USA. The Biorad Protein Assay Reagent Concentrate was purchased from Biorad Laboratories (Hercules, CA). Thiophene-2-thiolate was obtained from Alfa Aesar MA, USA. Iodine was obtained from Mallinckrodt Kentucky, USA, and acetonitrile was obtained from EMD Chemicals (New Jersey, USA). Sodium phosphate monohydrate and potassium phosphate dihydrate were acquired from EM Science (New Jersey, USA). Sodium carbonate was obtained from Orion Research Inc. (Beverly, USA). Magnetic nanoparticles (Fe 3 O 4 ) were prepared by chemical co-precipitation of Fe 2+  and Fe 3+  ions in a solution of ammonium hydroxide (magnetic nanoparticles I or Fe 3 O 4  I), or sodium hydroxide (magnetic nanoparticles II or Fe 3 O 4  II) followed by a treatment under hydrothermal conditions [ 4 , 5 ]. Iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25°C) by adding NH 4 OH solution (30%) or NaOH 3 M at a pH 10. The precipitates were heated at 80°C for 35 min under continuous mixing and washed 4 times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the supernatant using a magnetic separator of strength greater than 20 megaoersted (MOe). The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction. Magnetic nanoparticles I (50 mg) produced, using a solution of ammonium hydroxide were added to 1 mL of phosphate buffer (0.05 M. pH 7.4). After adding 1 mL of carbodiimide solution (0.02 g/mL) in phosphate buffer (0.05 M. pH 7.4), the mixture was sonicated for 15 min. Following the carbodiimide activation, 2 mL of glucose oxidase (1000 units /mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath. The magnetic nanoparticles were separated from the mixture using a magnetic separator. The precipitates containing Fe 3 O 4  nanoparticles I and Fe 3 O 4  bound glucose oxidase (GOX-Fe 3 O 4  I) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles [ 4 ]. A second functionalization protocol using a modification of the strategy adopted to immobilize  Candida rugosa  lipase on the γ-Fe 2 O 3 [ 12 ] was implemented (Magnetic nanoparticles II). Briefly, 1.5 g Fe 3 O 4  was added to 5 g of 11-bromoundecanoic acid dissolved in 15 mL of ethanol. 11-bromoundecanoic acid was covalently linked to the nanoparticles surfaces by heating the mixture with microwave irradiation for 10 min. Functionalization of the particle was achieved through nucleophilic substitution with the 2-thiophene thiolate. In practice, 2-thiophene thiolate (7 mL) was added to the mixture containing the particles and heated in microwave for 5 minutes. The mixture was washed with ethanol and transferred in a round bottom flask. Acetic anhydride (4 mL) and 34.6 mL of iodine (0.01N) were successively added to the particles and agitated. The mixture was heated for 1 h under reflux condition [ 17 ]. The particles were washed several times with water, once with 10% sodium carbonate solution and finally with ethanol. The acetylated particles were reacted directly with the enzyme covalently linked to the particles via C = N bond [ 11 ]. For the attachment of glucose to nanoparticles, 2 mL of the GOX solution (1000 units/mL) was added to 50 mg of functionalized magnetic nanoparticles in a test tube and sonicated at 15°C for 3 h. The supernatant containing unbound enzymes was separated from the magnetic nanoparticles using the magnetic separator, and the enzymes bound to magnetic nanoparticles were then used for activity determination. A schematic of the procedures used for both attachments are presented in Figure  1 . The amount of protein in the supernatant was determined by a colorimetric method at 595 nm with the Biorad Protein Assay Reagent Concentrate using bovine serum albumin (BSA) as the protein standard. The amount of bound enzymes was calculated from: Figure 1 Description of GOX attachment procedures. (I) procedure employed for GOX-Fe 3 O 4  I attachment, (II) thiophene functionalization and the acetylation of particles for GOX-Fe 3 O 4  II attachment. A  = ( C i  -  C s )*  V      (1) Where  A  is the amount of bound enzyme,  C i  and  C s  are the concentration of the enzyme initially added for attachment and in the supernatant, respectively (mg/mL) and  V  is the volume of the reaction medium (mL). The size of Fe 3 O 4  magnetic nanoparticles, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II were characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL) and structure by FTIR spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared as follows: a drop of magnetic nanoparticles was dispersed in nanopure water. The resulting solution was sonicated for 4 min to obtain better particle dispersion. A drop of the dispersed solution was then deposited onto a copper grid and dried overnight at room temperature. The binding of GOX onto the magnetic nanoparticles was investigated using FTIR spectroscopy. Samples for FTIR analysis were prepared in phosphate buffer pH 7.4. The activity of bound GOX was determined by measuring the initial rate of formation of hydrogen peroxide at a given temperature following the formation of a red quinoneimine dye. The principle of enzymatic determination of the activity of glucose oxidase is described as follows: Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide. Phenol + 4-AAP, in the presence of peroxidase (POX), produces a quinoneimine dye that is measured at 500 nm using a Beckman Du Spectrometer to provide an absorbance that is proportional to the concentration of glucose in the sample. The reaction is described as follows: The activity of glucose oxidase was measured as follows. An assay mixture was prepared by mixing 500 U of horseradish peroxidase, 0.015 mmol of 4-aminoantipyrine (4-AAP), 0.025 mmol of phenol and 5 mmol of glucose in 50 mL of phosphate buffer solution (0.05 M. pH 7.4) to result in a glucose concentration of 0.1 M. To start the enzymatic reaction, 2 mL of the assay solution was added to 15 mL centrifuge test tubes containing GOX-Fe 3 O 4  and mixed by vortex. A solution of free GOX of the same molar concentration was used to evaluate the activity of the free enzyme for comparison. The solution was incubated at various temperatures (37–80°C) at specific intervals of time (30 min) and the supernatant was separated from GOX-Fe 3 O 4  using a magnetic separator. 10 μL aliquots of the supernatant were then taken for determining the concentration of hydrogen peroxide following the procedure by Trinder [ 18 ]. The activity of the enzyme can be calculated using the following equation: Where  ABS  sample denotes the absorbance of the sample,  ABS Std  is the absorbance of the standard solution, and C the concentration of glucose in the sample. The effect of temperature on the free GOX, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II was estimated by determining the concentration of glucose in the sample at various temperatures. A solution of the assay mixture was added to the various centrifuge test tubes containing bound or free enzymes. The samples were stored in a water bath at specific temperatures (37, 50, 60, 70 and 80°C) and the absorbance was monitored at fixed time intervals to determine the glucose content. The effect of pH on GOX was monitored by measuring the initial rate of glucose oxidation by glucose oxidase in different phosphate and carbonate buffer solutions of pH (5–10) at 25°C. The thermal stability of free GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (37–80°C) in phosphate buffer (0.05 M, pH 7.4) for 30 min. Aliquots of the reacting solutions were taken at periodic intervals (every 30 min for 6 h) and assayed for enzymatic activity as described above. The first-order inactivation rate constant,  k  was calculated from the equation: ln  A  = ln  A 0  -  kt      (5) where  A 0  is the initial activity,  A  is the activity after time t (min) and  k  is the reaction constant. The storage stability was examined by measuring the change in the concentration of glucose at room temperature at different time intervals (4 days). Test tubes with samples of GOX-Fe 3 O 4  I, GOX-Fe 3 O 4  II, or free GOX solutions were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 33 days. Thereafter, 3 mL of the assay solution was added, and the residual activity of GOX was assayed. The kinetic parameters of free GOX, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II,  K m  and  V max  were determined by measuring the initial rates of glucose oxidation (0.2–1 mM) by glucose oxidase (0.25 mg/mL) in phosphate buffer (pH 7.4) at 25°C.
Materials and methods
Glucose oxidase (specific activity 200 units/mg protein) from  Aspergillus  niger was purchased from VWR international (Pittsburgh, USA). Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide, sodium hydroxide, acetic anhydride, glucose, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 % and iron (III) chloride hexahydrate 99%, were obtained from Sigma-Aldrich St Louis (USA). 11-bromoundecanoic acid was obtained from TCI America Portland, USA. The Biorad Protein Assay Reagent Concentrate was purchased from Biorad Laboratories (Hercules, CA). Thiophene-2-thiolate was obtained from Alfa Aesar MA, USA. Iodine was obtained from Mallinckrodt Kentucky, USA, and acetonitrile was obtained from EMD Chemicals (New Jersey, USA). Sodium phosphate monohydrate and potassium phosphate dihydrate were acquired from EM Science (New Jersey, USA). Sodium carbonate was obtained from Orion Research Inc. (Beverly, USA). Magnetic nanoparticles (Fe 3 O 4 ) were prepared by chemical co-precipitation of Fe 2+  and Fe 3+  ions in a solution of ammonium hydroxide (magnetic nanoparticles I or Fe 3 O 4  I), or sodium hydroxide (magnetic nanoparticles II or Fe 3 O 4  II) followed by a treatment under hydrothermal conditions [ 4 , 5 ]. Iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25°C) by adding NH 4 OH solution (30%) or NaOH 3 M at a pH 10. The precipitates were heated at 80°C for 35 min under continuous mixing and washed 4 times in water and several times in ethanol. During washing, the magnetic nanoparticles were separated from the supernatant using a magnetic separator of strength greater than 20 megaoersted (MOe). The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction.AspergillusMagnetic nanoparticles I (50 mg) produced, using a solution of ammonium hydroxide were added to 1 mL of phosphate buffer (0.05 M. pH 7.4). After adding 1 mL of carbodiimide solution (0.02 g/mL) in phosphate buffer (0.05 M. pH 7.4), the mixture was sonicated for 15 min. Following the carbodiimide activation, 2 mL of glucose oxidase (1000 units /mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath. The magnetic nanoparticles were separated from the mixture using a magnetic separator. The precipitates containing Fe 3 O 4  nanoparticles I and Fe 3 O 4  bound glucose oxidase (GOX-Fe 3 O 4  I) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles [ 4 ].A second functionalization protocol using a modification of the strategy adopted to immobilize  Candida rugosa  lipase on the γ-Fe 2 O 3 [ 12 ] was implemented (Magnetic nanoparticles II). Briefly, 1.5 g Fe 3 O 4  was added to 5 g of 11-bromoundecanoic acid dissolved in 15 mL of ethanol. 11-bromoundecanoic acid was covalently linked to the nanoparticles surfaces by heating the mixture with microwave irradiation for 10 min. Functionalization of the particle was achieved through nucleophilic substitution with the 2-thiophene thiolate. In practice, 2-thiophene thiolate (7 mL) was added to the mixture containing the particles and heated in microwave for 5 minutes. The mixture was washed with ethanol and transferred in a round bottom flask. Acetic anhydride (4 mL) and 34.6 mL of iodine (0.01N) were successively added to the particles and agitated. The mixture was heated for 1 h under reflux condition [ 17 ]. The particles were washed several times with water, once with 10% sodium carbonate solution and finally with ethanol. The acetylated particles were reacted directly with the enzyme covalently linked to the particles via C = N bond [ 11 ].Candida rugosaFor the attachment of glucose to nanoparticles, 2 mL of the GOX solution (1000 units/mL) was added to 50 mg of functionalized magnetic nanoparticles in a test tube and sonicated at 15°C for 3 h. The supernatant containing unbound enzymes was separated from the magnetic nanoparticles using the magnetic separator, and the enzymes bound to magnetic nanoparticles were then used for activity determination. A schematic of the procedures used for both attachments are presented in Figure  1 . The amount of protein in the supernatant was determined by a colorimetric method at 595 nm with the Biorad Protein Assay Reagent Concentrate using bovine serum albumin (BSA) as the protein standard. The amount of bound enzymes was calculated from:A  = ( C i  -  C s )*  V      (1)ACiCsVWhere  A  is the amount of bound enzyme,  C i  and  C s  are the concentration of the enzyme initially added for attachment and in the supernatant, respectively (mg/mL) and  V  is the volume of the reaction medium (mL). The size of Fe 3 O 4  magnetic nanoparticles, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II were characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL) and structure by FTIR spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared as follows: a drop of magnetic nanoparticles was dispersed in nanopure water. The resulting solution was sonicated for 4 min to obtain better particle dispersion. A drop of the dispersed solution was then deposited onto a copper grid and dried overnight at room temperature. The binding of GOX onto the magnetic nanoparticles was investigated using FTIR spectroscopy. Samples for FTIR analysis were prepared in phosphate buffer pH 7.4. The activity of bound GOX was determined by measuring the initial rate of formation of hydrogen peroxide at a given temperature following the formation of a red quinoneimine dye. The principle of enzymatic determination of the activity of glucose oxidase is described as follows: Glucose is oxidized by glucose oxidase to gluconate and hydrogen peroxide. Phenol + 4-AAP, in the presence of peroxidase (POX), produces a quinoneimine dye that is measured at 500 nm using a Beckman Du Spectrometer to provide an absorbance that is proportional to the concentration of glucose in the sample. The reaction is described as follows:ACiCsVThe activity of glucose oxidase was measured as follows. An assay mixture was prepared by mixing 500 U of horseradish peroxidase, 0.015 mmol of 4-aminoantipyrine (4-AAP), 0.025 mmol of phenol and 5 mmol of glucose in 50 mL of phosphate buffer solution (0.05 M. pH 7.4) to result in a glucose concentration of 0.1 M. To start the enzymatic reaction, 2 mL of the assay solution was added to 15 mL centrifuge test tubes containing GOX-Fe 3 O 4  and mixed by vortex. A solution of free GOX of the same molar concentration was used to evaluate the activity of the free enzyme for comparison. The solution was incubated at various temperatures (37–80°C) at specific intervals of time (30 min) and the supernatant was separated from GOX-Fe 3 O 4  using a magnetic separator. 10 μL aliquots of the supernatant were then taken for determining the concentration of hydrogen peroxide following the procedure by Trinder [ 18 ]. The activity of the enzyme can be calculated using the following equation:Where  ABS  sample denotes the absorbance of the sample,  ABS Std  is the absorbance of the standard solution, and C the concentration of glucose in the sample.ABSABS StdThe effect of temperature on the free GOX, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II was estimated by determining the concentration of glucose in the sample at various temperatures. A solution of the assay mixture was added to the various centrifuge test tubes containing bound or free enzymes. The samples were stored in a water bath at specific temperatures (37, 50, 60, 70 and 80°C) and the absorbance was monitored at fixed time intervals to determine the glucose content. The effect of pH on GOX was monitored by measuring the initial rate of glucose oxidation by glucose oxidase in different phosphate and carbonate buffer solutions of pH (5–10) at 25°C.The thermal stability of free GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (37–80°C) in phosphate buffer (0.05 M, pH 7.4) for 30 min. Aliquots of the reacting solutions were taken at periodic intervals (every 30 min for 6 h) and assayed for enzymatic activity as described above. The first-order inactivation rate constant,  k  was calculated from the equation:kln  A  = ln  A 0  -  kt      (5)AAktwhere  A 0  is the initial activity,  A  is the activity after time t (min) and  k  is the reaction constant.AAkThe storage stability was examined by measuring the change in the concentration of glucose at room temperature at different time intervals (4 days). Test tubes with samples of GOX-Fe 3 O 4  I, GOX-Fe 3 O 4  II, or free GOX solutions were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 33 days. Thereafter, 3 mL of the assay solution was added, and the residual activity of GOX was assayed.The kinetic parameters of free GOX, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II,  K m  and  V max  were determined by measuring the initial rates of glucose oxidation (0.2–1 mM) by glucose oxidase (0.25 mg/mL) in phosphate buffer (pH 7.4) at 25°C.KVResults and discussion Synthesis of magnetic nanoparticles with equivalent amounts of ferric and ferrous chlorides resulted in 66% of magnetic nanoparticles I and 73% of magnetic nanoparticles II. These yields suggested that the synthesis using NaOH is more advantageous for large scale production of magnetic nanoparticles. "Bare" Fe 3 O 4  I and Fe 3 O 4  II, and their GOX bound counterparts shown in the TEM micrographs in Figures ( 2A, B, C  and  2D ), respectively reveal that the particles are fine and spherical. The sizes of the particles of each sample were evaluated from 2 different TEM images. The diameter was in the range between 9 and 13 nm. The coefficient of variation between different measurements was less than 7%. There was no significant change in the size of the 'bare" particles and GOX bound particles. However, signs of agglomeration of the particles were visible in the samples. The agglomerates were not considered in the examination of the size distribution of the magnetic particles because of the assumption that agglomerated particles do not describe the original size of the particles. Figure  3  shows the distribution of the particles sizes. Fe 3 O 4 nanoparticles 14.5 mg/mL corresponding to GOX/Fe 3 O 4  weight ratios of 0.2 was used for the binding process. The amount of unbound enzyme was determined by assaying the protein content in the supernatant. The amount of enzymes bound to the magnetic nanoparticles in each binding procedure is given in table  1 . Average binding efficiencies of 38.4 units/mg particles for GOX-Fe 3 O 4  I and 27.6 units/mg particles for GOX-Fe 3 O 4  II were noted. The overall percentage of binding efficiency was between 94 and 100% for GOX-Fe 3 O 4  I and 66 to 72% for GOX-Fe 3 O 4  II. These results show that the binding was successful in both cases, particularly with Fe 3 O 4  I which uses the carbodiimide activation. The percentage of binding of GOX to Fe 3 O 4 II was significant but still below the level obtained through carbodiimide activation with the Fe 3 O 4  I. However, despite the difference in the level of GOX bound, the two procedures adopted were successful. Figure 2 Transmission electron micrographs of Fe 3 O 4  magnetic nanoparticles I (A), Fe 3 O 4  magnetic nanoparticles II (B), GOX-Fe 3 O 4  I (C), and GOX-Fe 3 O 4  II (D). Figure 3 Distribution of particles on the electron micrographs of Fe 3 O 4  magnetic nanoparticles I and GOX-Fe 3 O 4  I (A), and Fe 3 O 4  magnetic nanoparticles II and GOX-Fe 3 O 4  II (B). Table 1 Summary of binding efficiencies of enzyme-functionalized systems (n = 9). Bound enzymes (units/mg nanoparticles) GOX-Fe 3 O 4  I 38.4 ± 0.8 GOX-Fe 3 O 4  II 27.6 ± 0.6 The FTIR spectra of magnetic Fe 3 O 4  I, Fe 3 O 4  II, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II are shown in Figure  4(A, B, C, D) , respectively. The characteristic bands of proteins at 1541 and 1645 cm -1  assigned to amide I and amide II, respectively were visible in the spectra of GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II. These peaks are associated with two sharp peaks in the region 1420-1300 cm -1  typical of carboxylate groups, from the enzyme. A weak peak at 1900 cm -1  appeared in the spectra of GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II and could be assigned to the C-O bonds in the enzyme molecule. These peaks were absent in the spectra of Fe 3 O 4  I and Fe 3 O 4  II. In the 1100-1000 cm -1  region in all spectra appeared a characteristic adsorption spectra typical to phosphate ion that can be assigned to the phosphate buffer used in the samples preparation. The occurrence of negative peaks in the spectra of Fe 3 O 4  I, and GOX-Fe 3 O 4  II is possibly due to the reduced amount of phosphate in comparison to the amount of phosphate used to background subtraction. The characteristic peaks of proteins found in the spectra of GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II were not visible in the spectra of Fe 3 O 4  I and Fe 3 O 4  II, indicating enzyme attachment onto the particles. The amino groups on the surface of the particles resulted from the use of concentrated ammonia solution during the co-precipitation of Fe 2+  and Fe 3+  as demonstrated by [ 5 ] and [ 19 ]. Figure 4 FTIR spectra of Fe 3 O 4  magnetic nanoparticles I (A) and Fe 3 O 4  magnetic nanoparticles II (B), GOX-Fe 3 O 4  I (C), and GOX-Fe 3 O 4  II (D). The most important aspect of this study is related to the retention of biocatalytical activity of GOX after binding to magnetic nanoparticles. The amount of bound GOX was estimated using the UV-vis spectrophotometer and the catalytic activity of free and bound GOX was compared. Kinetic parameters ( V m  and  K m ) were estimated from the double reciprocal plots of the initial rates of glucose oxidation by GOX. The double reciprocal plots are presented in Figure  5 . The Michaelis-Menten constants  V max  and  K m  for GOX are shown in Table  2 .  V max  of free GOX, GOX- Fe 3 O 4  I and GOX-Fe 3 O 4  II were 0.731, 0.803, and 0766 μmol/min mL and, the corresponding  K m  values were, 0.383, 0.208, and 0.237 mM, respectively. The  V max  value of GOX immobilized on magnetic nanoparticles (I and II) was higher than that of the free enzyme. The highest  V max  was obtained with GOX-nanoparticles I. Since a low  K m  indicates a high degree of affinity of the enzyme to substrate [ 5 ], calculations showed that the affinity of the enzyme to the substrate increased in the order of free GOX, GOX-Fe 3 O 4  II and GOD-Fe 3 O 4  I, respectively. The high affinity of the enzyme to the substrate may be explained by a favorable change in the structural organization of the enzyme due to the immobilization procedure [ 20 ]. Consequently, the active sites of the enzymes could be more readily available for enzymatic interactions. Figure 5 Double reciprocal plots of the initial rates of free GOX (■), GOX-Fe 3 O 4  I (▲) and GOX-Fe 3 O 4  II (●) at pH 7.4, from experimental data. Table 2 Kinetic parameters of free GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II determined from the double reciprocal plots. V max  (μmol/min mL) K m  (mM) Free GOD 0.731 0.383 GOD-Fe 3 O 4  I 0.803 0.208 GOD-Fe 3 O 4  II 0.766 0.237 The effect of pH on the activities of the free and bound GOX was investigated in the pH range of 5–10 at 25°C (Figure  6 ). Each data point was the average of two measurements. The coefficient of variation between measurements was between 2 and 5%. In the pH range between 6 and 7.4, the enzyme activity increased in all the systems. However, the enzyme activity was higher in GOX-Fe 3 O 4  I than in GOX-Fe 3 O 4  II and free GOX. The activity reached 100% at pH 7.4, and decreased above this pH value to 43% for GOX-Fe 3 O 4  I, and to 26% for GOX-Fe 3 O 4  II and 9% for the free GOX at pH 10. The free GOX experienced a more severe loss in activity, while GOX-Fe 3 O 4  I retained greater activity as the pH increased. In this system, the binding process occurred directly upon activation of the particle surface using carbodiimide, while the binding of GOX-Fe 3 O 4  II involved a C = N bond formed on the acetylated thiophene. It can be argued that the direct binding between the protein and the amino bond in the former case exhibited a greater resistance to a medium with higher alkalinity. This medium appeared even more constraining to the free enzyme and placed the enzyme in an electrostatic state that might affect the activity. Figure 6 Effect of pH on the activities of free GOX (■), GOX-Fe 3 O 4  I (▲) and GOX-Fe 3 O 4  II (●). The effect of temperature on the activity of free GOX was examined by measuring its relative activity when stored at various temperatures. Figure  7a  and  7b  shows the effect of temperature on GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II at various temperatures. Each data point represents the average of duplicate measurements (coefficient of variation was less than 6 %). It can be observed that at 37°C, the enzyme retained its activity for about 80 minutes before showing a slight decrease. At 50, 60, 70 and 80°C the activity decreased as the temperature increased in both systems. In GOX- Fe 3 O 4 , the remaining activity was 23% at 50°C and 15% at 60°C after 270 min. For this time period and duration, the remaining activities were 9% and 0%, respectively for GOX-Fe 3 O 4  II. A similar trend was observed at 70 and 80°C with a more drastic loss of activity from the GOX-Fe 3 O 4  II protocol. Loss of activity occurred more rapidly in GOX-Fe 3 O 4  II indicating that direct binding through carbodiimide activation provides a greater thermal stability to GOX. The effect of temperature on the activities of free, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II at pH 7.4 are presented in the Arrhenius plots (Figure  8 ). The activation energies were calculated as 1.4, 0.9, and 1.1 kJ/mol for free-GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II, respectively. These results show that the unbound enzyme has the highest activation energy, while GOX-Fe 3 O 4  I had the lowest. The low activation energy of GOX associated with binding to magnetic nanoparticles suggests that the energy requirement on the surfaces of the nanoparticles for enzymatic activity is reduced. Table  3  shows the inactivation rates constants ( k ) at 50, 60 70, and 80°C. The rate constants increased with increasing temperature in the order GOX-Fe 3 O 4  I, GOX-Fe 3 O 4  II and free GOX. Here again, binding GOX to magnetic nanoparticles minimized structural denaturation due to heat treatment. Covalent binding was expected to provide the enzyme with the protection against structural denaturation due to the unfavorable solvent-protein interactions, and thus result in activation effect [ 21 ], a possible reason for a better activity of the bound enzyme compared with the free enzyme after heat treatment. GOX-Fe 3 O 4  II had a lower stability at higher temperatures compared to GOX-Fe 3 O 4  I. The reason for this difference could reside in the stability of the binding, since the binding methods are so far the major difference between these systems. Indeed with GOX-Fe 3 O 4  I, the binding of the enzyme occurred through the amino groups on the surfaces of the particles and the carboxylic groups of proteins in the enzymes [ 5 ] which is a natural way for protein binding, while in the case of GOX-Fe 3 O 4  II the N atom to which the enzyme is attached shared a double bond with the carbon atom which is less stable than the amide bond. Figure 7 Effect of temperature on the activity of GOX-Fe 3 O 4  I (A) and GOX-Fe 3 O 4  II (B) at pH 7.4. The samples were stored at 37, 50, 60, 70 and 80°C for 30 min and the activities were measured at 25°C. Figure 8 Arrhenius plots of the initial oxidation rates of glucose by free GOX (■), GOX-Fe 3 O 4  I (▲) and GOX-Fe 3 O 4  II (●) for samples at 37, 50, 60, 70, and 80°C. Table 3 Inactivation rate constants ( k ) of the free-GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4 II at various temperatures. Temperature (°C) Free-GOX  k  (min -1 ) GOX-Fe 3 O 4  I  k  (min -1 ) GOX-Fe 3 O 4  II  k  (min -1 ) 50 3.42. 10 -2 7.10.10 -4 2.87.10 -3 60 9.36.10 -2 1.49.10 -3 3.50.10 -2 70 2.81.10 -1 6.24.10 -2 1.92.10 -2 80 9.3210 -1 4.52.10 -2 5.46.10 -1 Loss of storage stability is a major concern in enzyme preservation. The storage stability of the enzyme was examined for 33 days. Figure  9  shows the storage stabilities of free GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II at 25°C at pH 7.4. Each data point was the average of duplicate measurements (coefficient of variation of the measurements was between 1 and 5%). The activity decreased with time in all the systems. Total loss of activity was observed after 20 days for the free GOX and 28 days for GOX-Fe 3 O 4  II while GOX-Fe 3 O 4  I retained 26% activity after 33 days of storage under identical conditions. The stability of the enzyme was found to improve upon binding to the magnetic nanoparticles but the most significant improvement in stability was observed for the GOX-Fe 3 O 4  I nanoparticle complex. The fixation on the surface of the magnetic nanoparticles has been a tangible argument supporting the prevention of auto-digestion of the enzyme and lysis, and the subsequent conservation of its activity [ 4 ]. This argument supports our results and justifies the long term stability of GOX-nanoparticles I and GOX-nanoparticles II over the free GOX. The efficiency of binding of GOX via carbodiimide activation over the binding by thiophene acetylation may be attributed to the potential of carbodiimide to activate the carboxylic acid side chains partially buried at the surface or in active sites of the enzyme, as well as the amino groups on the nanoparticles, favoring the formation and the stability of the amide bond [ 22 ]. This may explain why the amount of bound enzymes is higher in the binding via carbodiimide than with the thiophene acetylation. Furthermore, carbodiimide might cause cross-linking of the enzyme providing a better stability to its quaternary structure [ 23 ] and a subsequent improvement in stability. Figure 9 Storage stability of free GOX (■), GOX-Fe 3 O 4  I (▲), and GOX-Fe 3 O 4  II (●) measured at a pH of 7.4 at 25°C.
Results and discussion
Synthesis of magnetic nanoparticles with equivalent amounts of ferric and ferrous chlorides resulted in 66% of magnetic nanoparticles I and 73% of magnetic nanoparticles II. These yields suggested that the synthesis using NaOH is more advantageous for large scale production of magnetic nanoparticles. "Bare" Fe 3 O 4  I and Fe 3 O 4  II, and their GOX bound counterparts shown in the TEM micrographs in Figures ( 2A, B, C  and  2D ), respectively reveal that the particles are fine and spherical. The sizes of the particles of each sample were evaluated from 2 different TEM images. The diameter was in the range between 9 and 13 nm. The coefficient of variation between different measurements was less than 7%. There was no significant change in the size of the 'bare" particles and GOX bound particles. However, signs of agglomeration of the particles were visible in the samples. The agglomerates were not considered in the examination of the size distribution of the magnetic particles because of the assumption that agglomerated particles do not describe the original size of the particles. Figure  3  shows the distribution of the particles sizes. Fe 3 O 4 nanoparticles 14.5 mg/mL corresponding to GOX/Fe 3 O 4  weight ratios of 0.2 was used for the binding process. The amount of unbound enzyme was determined by assaying the protein content in the supernatant. The amount of enzymes bound to the magnetic nanoparticles in each binding procedure is given in table  1 . Average binding efficiencies of 38.4 units/mg particles for GOX-Fe 3 O 4  I and 27.6 units/mg particles for GOX-Fe 3 O 4  II were noted. The overall percentage of binding efficiency was between 94 and 100% for GOX-Fe 3 O 4  I and 66 to 72% for GOX-Fe 3 O 4  II. These results show that the binding was successful in both cases, particularly with Fe 3 O 4  I which uses the carbodiimide activation. The percentage of binding of GOX to Fe 3 O 4 II was significant but still below the level obtained through carbodiimide activation with the Fe 3 O 4  I. However, despite the difference in the level of GOX bound, the two procedures adopted were successful.Summary of binding efficiencies of enzyme-functionalized systems (n = 9).The FTIR spectra of magnetic Fe 3 O 4  I, Fe 3 O 4  II, GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II are shown in Figure  4(A, B, C, D) , respectively. The characteristic bands of proteins at 1541 and 1645 cm -1  assigned to amide I and amide II, respectively were visible in the spectra of GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II. These peaks are associated with two sharp peaks in the region 1420-1300 cm -1  typical of carboxylate groups, from the enzyme. A weak peak at 1900 cm -1  appeared in the spectra of GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II and could be assigned to the C-O bonds in the enzyme molecule. These peaks were absent in the spectra of Fe 3 O 4  I and Fe 3 O 4  II. In the 1100-1000 cm -1  region in all spectra appeared a characteristic adsorption spectra typical to phosphate ion that can be assigned to the phosphate buffer used in the samples preparation. The occurrence of negative peaks in the spectra of Fe 3 O 4  I, and GOX-Fe 3 O 4  II is possibly due to the reduced amount of phosphate in comparison to the amount of phosphate used to background subtraction. The characteristic peaks of proteins found in the spectra of GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II were not visible in the spectra of Fe 3 O 4  I and Fe 3 O 4  II, indicating enzyme attachment onto the particles. The amino groups on the surface of the particles resulted from the use of concentrated ammonia solution during the co-precipitation of Fe 2+  and Fe 3+  as demonstrated by [ 5 ] and [ 19 ].The most important aspect of this study is related to the retention of biocatalytical activity of GOX after binding to magnetic nanoparticles. The amount of bound GOX was estimated using the UV-vis spectrophotometer and the catalytic activity of free and bound GOX was compared. Kinetic parameters ( V m  and  K m ) were estimated from the double reciprocal plots of the initial rates of glucose oxidation by GOX. The double reciprocal plots are presented in Figure  5 . The Michaelis-Menten constants  V max  and  K m  for GOX are shown in Table  2 .  V max  of free GOX, GOX- Fe 3 O 4  I and GOX-Fe 3 O 4  II were 0.731, 0.803, and 0766 μmol/min mL and, the corresponding  K m  values were, 0.383, 0.208, and 0.237 mM, respectively. The  V max  value of GOX immobilized on magnetic nanoparticles (I and II) was higher than that of the free enzyme. The highest  V max  was obtained with GOX-nanoparticles I. Since a low  K m  indicates a high degree of affinity of the enzyme to substrate [ 5 ], calculations showed that the affinity of the enzyme to the substrate increased in the order of free GOX, GOX-Fe 3 O 4  II and GOD-Fe 3 O 4  I, respectively. The high affinity of the enzyme to the substrate may be explained by a favorable change in the structural organization of the enzyme due to the immobilization procedure [ 20 ]. Consequently, the active sites of the enzymes could be more readily available for enzymatic interactions.VmKmVKVKVVKKinetic parameters of free GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II determined from the double reciprocal plots.KThe effect of pH on the activities of the free and bound GOX was investigated in the pH range of 5–10 at 25°C (Figure  6 ). Each data point was the average of two measurements. The coefficient of variation between measurements was between 2 and 5%. In the pH range between 6 and 7.4, the enzyme activity increased in all the systems. However, the enzyme activity was higher in GOX-Fe 3 O 4  I than in GOX-Fe 3 O 4  II and free GOX. The activity reached 100% at pH 7.4, and decreased above this pH value to 43% for GOX-Fe 3 O 4  I, and to 26% for GOX-Fe 3 O 4  II and 9% for the free GOX at pH 10. The free GOX experienced a more severe loss in activity, while GOX-Fe 3 O 4  I retained greater activity as the pH increased. In this system, the binding process occurred directly upon activation of the particle surface using carbodiimide, while the binding of GOX-Fe 3 O 4  II involved a C = N bond formed on the acetylated thiophene. It can be argued that the direct binding between the protein and the amino bond in the former case exhibited a greater resistance to a medium with higher alkalinity. This medium appeared even more constraining to the free enzyme and placed the enzyme in an electrostatic state that might affect the activity.The effect of temperature on the activity of free GOX was examined by measuring its relative activity when stored at various temperatures. Figure  7a  and  7b  shows the effect of temperature on GOX-Fe 3 O 4  I and GOX-Fe 3 O 4  II at various temperatures. Each data point represents the average of duplicate measurements (coefficient of variation was less than 6 %). It can be observed that at 37°C, the enzyme retained its activity for about 80 minutes before showing a slight decrease. At 50, 60, 70 and 80°C the activity decreased as the temperature increased in both systems. In GOX- Fe 3 O 4 , the remaining activity was 23% at 50°C and 15% at 60°C after 270 min. For this time period and duration, the remaining activities were 9% and 0%, respectively for GOX-Fe 3 O 4  II. A similar trend was observed at 70 and 80°C with a more drastic loss of activity from the GOX-Fe 3 O 4  II protocol. Loss of activity occurred more rapidly in GOX-Fe 3 O 4  II indicating that direct binding through carbodiimide activation provides a greater thermal stability to GOX. The effect of temperature on the activities of free, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II at pH 7.4 are presented in the Arrhenius plots (Figure  8 ). The activation energies were calculated as 1.4, 0.9, and 1.1 kJ/mol for free-GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II, respectively. These results show that the unbound enzyme has the highest activation energy, while GOX-Fe 3 O 4  I had the lowest. The low activation energy of GOX associated with binding to magnetic nanoparticles suggests that the energy requirement on the surfaces of the nanoparticles for enzymatic activity is reduced. Table  3  shows the inactivation rates constants ( k ) at 50, 60 70, and 80°C. The rate constants increased with increasing temperature in the order GOX-Fe 3 O 4  I, GOX-Fe 3 O 4  II and free GOX. Here again, binding GOX to magnetic nanoparticles minimized structural denaturation due to heat treatment. Covalent binding was expected to provide the enzyme with the protection against structural denaturation due to the unfavorable solvent-protein interactions, and thus result in activation effect [ 21 ], a possible reason for a better activity of the bound enzyme compared with the free enzyme after heat treatment. GOX-Fe 3 O 4  II had a lower stability at higher temperatures compared to GOX-Fe 3 O 4  I. The reason for this difference could reside in the stability of the binding, since the binding methods are so far the major difference between these systems. Indeed with GOX-Fe 3 O 4  I, the binding of the enzyme occurred through the amino groups on the surfaces of the particles and the carboxylic groups of proteins in the enzymes [ 5 ] which is a natural way for protein binding, while in the case of GOX-Fe 3 O 4  II the N atom to which the enzyme is attached shared a double bond with the carbon atom which is less stable than the amide bond.kInactivation rate constants ( k ) of the free-GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4 II at various temperatures.kkkkLoss of storage stability is a major concern in enzyme preservation. The storage stability of the enzyme was examined for 33 days. Figure  9  shows the storage stabilities of free GOX, GOX-Fe 3 O 4  I, and GOX-Fe 3 O 4  II at 25°C at pH 7.4. Each data point was the average of duplicate measurements (coefficient of variation of the measurements was between 1 and 5%). The activity decreased with time in all the systems. Total loss of activity was observed after 20 days for the free GOX and 28 days for GOX-Fe 3 O 4  II while GOX-Fe 3 O 4  I retained 26% activity after 33 days of storage under identical conditions. The stability of the enzyme was found to improve upon binding to the magnetic nanoparticles but the most significant improvement in stability was observed for the GOX-Fe 3 O 4  I nanoparticle complex. The fixation on the surface of the magnetic nanoparticles has been a tangible argument supporting the prevention of auto-digestion of the enzyme and lysis, and the subsequent conservation of its activity [ 4 ]. This argument supports our results and justifies the long term stability of GOX-nanoparticles I and GOX-nanoparticles II over the free GOX. The efficiency of binding of GOX via carbodiimide activation over the binding by thiophene acetylation may be attributed to the potential of carbodiimide to activate the carboxylic acid side chains partially buried at the surface or in active sites of the enzyme, as well as the amino groups on the nanoparticles, favoring the formation and the stability of the amide bond [ 22 ]. This may explain why the amount of bound enzymes is higher in the binding via carbodiimide than with the thiophene acetylation. Furthermore, carbodiimide might cause cross-linking of the enzyme providing a better stability to its quaternary structure [ 23 ] and a subsequent improvement in stability.Conclusion Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides using two different base solutions. GOX was bound to the particles by direct attachment via carbodiimide activation and chemically via covalent attachment onto thiophene acetylated magnetic nanoparticles. Confirmation of the binding was demonstrated by FTIR spectroscopy and the sizes of the particles were characterized by TEM. The direct binding of GOX via carbodiimide activation was more effective and resulted in binding efficiency in the range between 94–100% while the binding efficiency was only between 66–72% for the GOX-Fe 3 O 4  II complex. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when the complex was subjected to thermal and pH variations. This study shows that binding onto magnetic nanoparticles can allow the enzyme to acquire the conformational and structural arrangement for a better activity and stability, and suggests that binding of enzyme onto magnetic nanoparticles via carbodiimide activation was efficient for creating bioconjugates for a variety of applications in health and food safety.
Conclusion
Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides using two different base solutions. GOX was bound to the particles by direct attachment via carbodiimide activation and chemically via covalent attachment onto thiophene acetylated magnetic nanoparticles. Confirmation of the binding was demonstrated by FTIR spectroscopy and the sizes of the particles were characterized by TEM. The direct binding of GOX via carbodiimide activation was more effective and resulted in binding efficiency in the range between 94–100% while the binding efficiency was only between 66–72% for the GOX-Fe 3 O 4  II complex. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when the complex was subjected to thermal and pH variations. This study shows that binding onto magnetic nanoparticles can allow the enzyme to acquire the conformational and structural arrangement for a better activity and stability, and suggests that binding of enzyme onto magnetic nanoparticles via carbodiimide activation was efficient for creating bioconjugates for a variety of applications in health and food safety.Authors' contributions Drs. Gilles Kouassi and Joseph Irudayaraj were the primary authors. They were responsible for the concept and experimental plan of the article. Dr Gregory MacCarty was the secondary author and contributed to the overall effort.
Authors' contributions
Drs. Gilles Kouassi and Joseph Irudayaraj were the primary authors. They were responsible for the concept and experimental plan of the article. Dr Gregory MacCarty was the secondary author and contributed to the overall effort.


Background Sentinel lymph node (SLN) biopsy is an effective tool for axillary staging in patients with invasive breast cancer. This procedure has been recently proposed as part of the treatment for patients with ductal carcinoma in situ (DCIS), because cases of undetected invasive foci and nodal metastases occasionally occur. However, the indications for SLN biopsy in DCIS patients are controversial. The aim of the present study was therefore to assess the incidence of SLN metastases in a series of patients with a diagnosis of pure DCIS.
Background
Sentinel lymph node (SLN) biopsy is an effective tool for axillary staging in patients with invasive breast cancer. This procedure has been recently proposed as part of the treatment for patients with ductal carcinoma in situ (DCIS), because cases of undetected invasive foci and nodal metastases occasionally occur. However, the indications for SLN biopsy in DCIS patients are controversial.The aim of the present study was therefore to assess the incidence of SLN metastases in a series of patients with a diagnosis of pure DCIS.Methods A retrospective evaluation was made of a series of 102 patients who underwent SLN biopsy, and had a final histologic diagnosis of pure DCIS. Patients with microinvasion were excluded from the analysis. The patients were operated on in five Institutions between 1999 and 2004. Subdermal or subareolar injection of 30–50 MBq of 99 m-Tc colloidal albumin was used for SLN identification. All sentinel nodes were evaluated with serial sectioning, haematoxylin and eosin staining, and immunohistochemical analysis for cytocheratin.
Methods
A retrospective evaluation was made of a series of 102 patients who underwent SLN biopsy, and had a final histologic diagnosis of pure DCIS. Patients with microinvasion were excluded from the analysis. The patients were operated on in five Institutions between 1999 and 2004.Subdermal or subareolar injection of 30–50 MBq of 99 m-Tc colloidal albumin was used for SLN identification. All sentinel nodes were evaluated with serial sectioning, haematoxylin and eosin staining, and immunohistochemical analysis for cytocheratin.Results Only one patient (0.98%) was SLN positive. The primary tumour was a small micropapillary intermediate-grade DCIS and the SLN harboured a micrometastasis. At pathologic revision of the specimen, no detectable focus of microinvasion was found.
Results
Only one patient (0.98%) was SLN positive. The primary tumour was a small micropapillary intermediate-grade DCIS and the SLN harboured a micrometastasis. At pathologic revision of the specimen, no detectable focus of microinvasion was found.Conclusion Our findings indicate that SLN metastases in pure DCIS are a very rare occurrence. SLN biopsy should not therefore be routinely performed in patients who undergo resection for DCIS. SLN mapping can be performed, as a second operation, in cases in which an invasive component is identified in the specimen. Only DCIS patients who require a mastectomy should have SLN biopsy performed at the time of breast operation, since in these cases subsequent node mapping is not feasible.
Conclusion
Our findings indicate that SLN metastases in pure DCIS are a very rare occurrence. SLN biopsy should not therefore be routinely performed in patients who undergo resection for DCIS. SLN mapping can be performed, as a second operation, in cases in which an invasive component is identified in the specimen. Only DCIS patients who require a mastectomy should have SLN biopsy performed at the time of breast operation, since in these cases subsequent node mapping is not feasible.

Background Ductal carcinoma in situ (DCIS) is defined as the proliferation of malignant epithelial cells in the mammary ductal system, with no evidence of invasion of the basement membrane. By definition, the disease is localized to the breast, with no spread to regional nodes or distant sites. In the pre-mammography era, DCIS was rarely diagnosed, accounting for only 1 to 2% of all breast cancers. The increasing use of screening mammography in recent years has resulted in a dramatic increase in the diagnosis of DCIS, which now accounts for roughly 20% of all mammographically detected cancers [ 1 ]. Axillary treatment in patients with a diagnosis of pure DCIS is controversial. By definition, pure DCIS is not invasive and cannot spread to the regional nodes, so axillary staging should not be indicated in DCIS patients. In practice, however, nodal metastases are found in 1 to 2% of patients with a diagnosis of pure DCIS, and this paradoxical condition usually depends on a focus of invasion being missed by the pathologist because of a sampling bias [ 2 ]. Before the introduction of sentinel lymph node (SLN) biopsy, it was widely agreed that axillary lymph node dissection (ALND) should be avoided in patients with DCIS, because of the low yield of positive findings in these cases and the significant morbidity associated with the procedure [ 3 ]. The introduction of SLN biopsy as a minimally invasive tool for staging the axilla led to a renewed interest in axillary staging for these patients, for two reasons. First, this procedure is far less invasive than ALND and has minimal morbidity and complications, so the cost/benefit ratio may be positive even if the likelihood of finding metastatic nodes is low. Second, SLN biopsy allows more accurate axillary staging than ALND specimen examination, because SLN can be thoroughly evaluated by serial sectioning and immunohistochemical methods. This has led to the increased detection of nodal micrometastases in patients with invasive breast cancer, and has raised the expectation that the rate of positive nodal findings in patients with DCIS could also be increased. Currently, the indications for SLN biopsy in patients with a diagnosis of pure DCIS are controversial. The aim of the present study was to determine the prevalence of SLN metastasis in a multi-institutional series of patients with a diagnosis of pure DCIS in order to determine the usefulness of routine SLN biopsy in such cases.
Background
Ductal carcinoma in situ (DCIS) is defined as the proliferation of malignant epithelial cells in the mammary ductal system, with no evidence of invasion of the basement membrane. By definition, the disease is localized to the breast, with no spread to regional nodes or distant sites.In the pre-mammography era, DCIS was rarely diagnosed, accounting for only 1 to 2% of all breast cancers. The increasing use of screening mammography in recent years has resulted in a dramatic increase in the diagnosis of DCIS, which now accounts for roughly 20% of all mammographically detected cancers [ 1 ].Axillary treatment in patients with a diagnosis of pure DCIS is controversial. By definition, pure DCIS is not invasive and cannot spread to the regional nodes, so axillary staging should not be indicated in DCIS patients. In practice, however, nodal metastases are found in 1 to 2% of patients with a diagnosis of pure DCIS, and this paradoxical condition usually depends on a focus of invasion being missed by the pathologist because of a sampling bias [ 2 ]. Before the introduction of sentinel lymph node (SLN) biopsy, it was widely agreed that axillary lymph node dissection (ALND) should be avoided in patients with DCIS, because of the low yield of positive findings in these cases and the significant morbidity associated with the procedure [ 3 ]. The introduction of SLN biopsy as a minimally invasive tool for staging the axilla led to a renewed interest in axillary staging for these patients, for two reasons. First, this procedure is far less invasive than ALND and has minimal morbidity and complications, so the cost/benefit ratio may be positive even if the likelihood of finding metastatic nodes is low. Second, SLN biopsy allows more accurate axillary staging than ALND specimen examination, because SLN can be thoroughly evaluated by serial sectioning and immunohistochemical methods. This has led to the increased detection of nodal micrometastases in patients with invasive breast cancer, and has raised the expectation that the rate of positive nodal findings in patients with DCIS could also be increased. Currently, the indications for SLN biopsy in patients with a diagnosis of pure DCIS are controversial.The aim of the present study was to determine the prevalence of SLN metastasis in a multi-institutional series of patients with a diagnosis of pure DCIS in order to determine the usefulness of routine SLN biopsy in such cases.Methods Patients Our series consisted of 102 patients with a final histopathological diagnosis of pure DCIS who underwent SLN biopsy in five Institutions between January 1999 and January 2004. Patients with DCIS with microinvasion were excluded from the study. All patients underwent SLN biopsy after they had given their written informed consent, and had decided whether they would undergo a completion ALND if SLN were found to be positive. Lymphoscintigraphy On the day before surgery, all the patients received an injection of 30–50 MBq of 99 m-Tc-nanocolloidal albumin (Nanocoll, Nicomed-Italia, Saluggia, Italy) in 0.2 cc of saline. In patients with palpable tumours, the radiocolloid was injected subdermally into the cutaneous projection of the tumour. In those with non-palpable tumours, the lesion was localized by stereotactic or ultrasonographic placement of a self-retaining wire and the cutaneous projection was marked by an ink spot on the skin, which served as a guide for subdermal radiocolloid injection. Patients with extensive microcalcifications or multifocal tumours were given subareolar radiotracer injection. Finally, patients who underwent delayed SLN biopsy after excision of the primary lesion were given subdermal radiotracer injection in two fractions, at the sides of the surgical scar. Twenty minutes and 2 hours after injection, scintigraphic images were obtained using a large-field-of-view gamma camera (Orbiter, Siemens, IL, USA) equipped with a parallel hole, low energy and a high resolution collimator. Surgical procedure Primary tumour resection with simultaneous SLN biopsy was performed in patients with a preoperative diagnosis of DCIS made by stereotactically-guided directional vacuum-assisted biopsy and in those with mammographical finding pathognomonic of DCIS, with or without a preoperative cytologic diagnosis of malignancy. A diagnostic excisional biopsy was performed in patients with a doubtful pre-operative diagnosis, and SLN biopsy was performed as a second operation in those with a final histologic diagnosis of DCIS. As a rule, in patients undergoing conservative surgery for non-palpable lesions, an intraoperative x-ray examination of the resected specimen was performed, in order to confirm that complete tumour excision had been achieved. SLN biopsy was performed 16 to 24 hours after radiocolloid injection. When conservative surgery was performed, the SLN was excised through the incision used for resecting the primary tumour, if located in the upper outer quadrant, whereas a separate axillary incision was performed if the tumour was located in another breast site. A gamma-ray-detecting probe was used for intraoperative SLN identification. All lymph nodes with a probe-detected radioactive count >10% of that of the hottest node were excised. Histopathology The excised breast lesions were sampled with serial cuts and the margins were identified by ink. The histopathologic diagnosis and classification of DCIS was made according to the Holland grading system [ 4 ]. The Van Nuys prognostic index [ 5 ] was used only in some of the cases. The search for microinvasive foci was performed in selected cases both with haematoxylin-eosin serial sections and immunostains to smooth muscle actin and CD10 for the detection of myoepithelial cells. In all cases, the tumour size and margin status were specified in the histopathological report. The SLN examination was performed as described elsewhere [ 6 ]. Briefly, for frozen-section examination, nodes with diameters of ≤ 0.5 cm were bisected, while nodes measuring >0.5 cm were sectioned each 2 to 3 mm. For each sample, two frozen sections made at 40 μm intervals were examined. The frozen tissue was then thawed, fixed and embedded to obtain permanent sections. For definitive histological examination, two consecutive 5 μm thick tissue sections were cut from a paraffin block at two levels, 40 μm apart from each other. The sections were then stained with haematoxylin-eosin and immunostained with monoclonal antibody to cytokeratin.
Methods
Patients Our series consisted of 102 patients with a final histopathological diagnosis of pure DCIS who underwent SLN biopsy in five Institutions between January 1999 and January 2004. Patients with DCIS with microinvasion were excluded from the study. All patients underwent SLN biopsy after they had given their written informed consent, and had decided whether they would undergo a completion ALND if SLN were found to be positive.
Patients
Our series consisted of 102 patients with a final histopathological diagnosis of pure DCIS who underwent SLN biopsy in five Institutions between January 1999 and January 2004. Patients with DCIS with microinvasion were excluded from the study.All patients underwent SLN biopsy after they had given their written informed consent, and had decided whether they would undergo a completion ALND if SLN were found to be positive.Lymphoscintigraphy On the day before surgery, all the patients received an injection of 30–50 MBq of 99 m-Tc-nanocolloidal albumin (Nanocoll, Nicomed-Italia, Saluggia, Italy) in 0.2 cc of saline. In patients with palpable tumours, the radiocolloid was injected subdermally into the cutaneous projection of the tumour. In those with non-palpable tumours, the lesion was localized by stereotactic or ultrasonographic placement of a self-retaining wire and the cutaneous projection was marked by an ink spot on the skin, which served as a guide for subdermal radiocolloid injection. Patients with extensive microcalcifications or multifocal tumours were given subareolar radiotracer injection. Finally, patients who underwent delayed SLN biopsy after excision of the primary lesion were given subdermal radiotracer injection in two fractions, at the sides of the surgical scar. Twenty minutes and 2 hours after injection, scintigraphic images were obtained using a large-field-of-view gamma camera (Orbiter, Siemens, IL, USA) equipped with a parallel hole, low energy and a high resolution collimator.
Lymphoscintigraphy
On the day before surgery, all the patients received an injection of 30–50 MBq of 99 m-Tc-nanocolloidal albumin (Nanocoll, Nicomed-Italia, Saluggia, Italy) in 0.2 cc of saline.In patients with palpable tumours, the radiocolloid was injected subdermally into the cutaneous projection of the tumour. In those with non-palpable tumours, the lesion was localized by stereotactic or ultrasonographic placement of a self-retaining wire and the cutaneous projection was marked by an ink spot on the skin, which served as a guide for subdermal radiocolloid injection. Patients with extensive microcalcifications or multifocal tumours were given subareolar radiotracer injection. Finally, patients who underwent delayed SLN biopsy after excision of the primary lesion were given subdermal radiotracer injection in two fractions, at the sides of the surgical scar.Twenty minutes and 2 hours after injection, scintigraphic images were obtained using a large-field-of-view gamma camera (Orbiter, Siemens, IL, USA) equipped with a parallel hole, low energy and a high resolution collimator.Surgical procedure Primary tumour resection with simultaneous SLN biopsy was performed in patients with a preoperative diagnosis of DCIS made by stereotactically-guided directional vacuum-assisted biopsy and in those with mammographical finding pathognomonic of DCIS, with or without a preoperative cytologic diagnosis of malignancy. A diagnostic excisional biopsy was performed in patients with a doubtful pre-operative diagnosis, and SLN biopsy was performed as a second operation in those with a final histologic diagnosis of DCIS. As a rule, in patients undergoing conservative surgery for non-palpable lesions, an intraoperative x-ray examination of the resected specimen was performed, in order to confirm that complete tumour excision had been achieved. SLN biopsy was performed 16 to 24 hours after radiocolloid injection. When conservative surgery was performed, the SLN was excised through the incision used for resecting the primary tumour, if located in the upper outer quadrant, whereas a separate axillary incision was performed if the tumour was located in another breast site. A gamma-ray-detecting probe was used for intraoperative SLN identification. All lymph nodes with a probe-detected radioactive count >10% of that of the hottest node were excised.
Surgical procedure
Primary tumour resection with simultaneous SLN biopsy was performed in patients with a preoperative diagnosis of DCIS made by stereotactically-guided directional vacuum-assisted biopsy and in those with mammographical finding pathognomonic of DCIS, with or without a preoperative cytologic diagnosis of malignancy. A diagnostic excisional biopsy was performed in patients with a doubtful pre-operative diagnosis, and SLN biopsy was performed as a second operation in those with a final histologic diagnosis of DCIS.As a rule, in patients undergoing conservative surgery for non-palpable lesions, an intraoperative x-ray examination of the resected specimen was performed, in order to confirm that complete tumour excision had been achieved.SLN biopsy was performed 16 to 24 hours after radiocolloid injection. When conservative surgery was performed, the SLN was excised through the incision used for resecting the primary tumour, if located in the upper outer quadrant, whereas a separate axillary incision was performed if the tumour was located in another breast site. A gamma-ray-detecting probe was used for intraoperative SLN identification. All lymph nodes with a probe-detected radioactive count >10% of that of the hottest node were excised.Histopathology The excised breast lesions were sampled with serial cuts and the margins were identified by ink. The histopathologic diagnosis and classification of DCIS was made according to the Holland grading system [ 4 ]. The Van Nuys prognostic index [ 5 ] was used only in some of the cases. The search for microinvasive foci was performed in selected cases both with haematoxylin-eosin serial sections and immunostains to smooth muscle actin and CD10 for the detection of myoepithelial cells. In all cases, the tumour size and margin status were specified in the histopathological report. The SLN examination was performed as described elsewhere [ 6 ]. Briefly, for frozen-section examination, nodes with diameters of ≤ 0.5 cm were bisected, while nodes measuring >0.5 cm were sectioned each 2 to 3 mm. For each sample, two frozen sections made at 40 μm intervals were examined. The frozen tissue was then thawed, fixed and embedded to obtain permanent sections. For definitive histological examination, two consecutive 5 μm thick tissue sections were cut from a paraffin block at two levels, 40 μm apart from each other. The sections were then stained with haematoxylin-eosin and immunostained with monoclonal antibody to cytokeratin.
Histopathology
The excised breast lesions were sampled with serial cuts and the margins were identified by ink.The histopathologic diagnosis and classification of DCIS was made according to the Holland grading system [ 4 ]. The Van Nuys prognostic index [ 5 ] was used only in some of the cases. The search for microinvasive foci was performed in selected cases both with haematoxylin-eosin serial sections and immunostains to smooth muscle actin and CD10 for the detection of myoepithelial cells. In all cases, the tumour size and margin status were specified in the histopathological report. The SLN examination was performed as described elsewhere [ 6 ]. Briefly, for frozen-section examination, nodes with diameters of ≤ 0.5 cm were bisected, while nodes measuring >0.5 cm were sectioned each 2 to 3 mm. For each sample, two frozen sections made at 40 μm intervals were examined. The frozen tissue was then thawed, fixed and embedded to obtain permanent sections.For definitive histological examination, two consecutive 5 μm thick tissue sections were cut from a paraffin block at two levels, 40 μm apart from each other. The sections were then stained with haematoxylin-eosin and immunostained with monoclonal antibody to cytokeratin.Results The 102 patients with pure DCIS had a median age of 59.4 years (range 37 to 85). The histologic characteristics of the tumours are reported in Table  1 . Of the 102 patients, 20 (19.6%) had palpable, and 82 (80.4%) non-palpable breast tumours. Seventy-four patients (72.5%) were treated with conservative surgery (quadrantectomy or lumpectomy) and 28 (27.5%) with mastectomy. Ninety-one patients underwent primary tumour resection with simultaneous SLN biopsy: 16 on the basis of a preoperative histologic diagnosis of DCIS obtained by a vacuum-assisted large core biopsy, 63 with a preoperative cytology suggestive of malignancy and 12 only on the basis of the mammographical findings alone. Patients with a doubtful preoperative diagnosis underwent excisional biopsy of the breast lesion, and the 11 patients with pure DCIS at definitive histology, underwent SLN biopsy and, if required, definitive treatment of the primary tumour as a second operation. A total of 147 SLNs were identified and excised from the 102 patients: a single SLN was found in 61 cases (59.8%), two SLNs in 37 (36.3%), and three SLNs in 4 (3.9%). A positive SLN was found in one (0.98%) patient, who had one micrometastases (0.6 mm) detected by haematoxylin-eosin staining. The pathologic review of the surgical specimen confirmed that the tumour had been completely excised, and no microinfiltration was detected. In this SLN positive case, the primary tumour was a micropapillary DCIS, G2, with a diameter of 16 mm. The patient did not undergone previous microbiopsy or FNAC, and SLN biopsy was performed at the time of the primary tumour excision. Completion ALND was not performed.
Results
The 102 patients with pure DCIS had a median age of 59.4 years (range 37 to 85). The histologic characteristics of the tumours are reported in Table  1 .Of the 102 patients, 20 (19.6%) had palpable, and 82 (80.4%) non-palpable breast tumours.Seventy-four patients (72.5%) were treated with conservative surgery (quadrantectomy or lumpectomy) and 28 (27.5%) with mastectomy.Ninety-one patients underwent primary tumour resection with simultaneous SLN biopsy: 16 on the basis of a preoperative histologic diagnosis of DCIS obtained by a vacuum-assisted large core biopsy, 63 with a preoperative cytology suggestive of malignancy and 12 only on the basis of the mammographical findings alone.Patients with a doubtful preoperative diagnosis underwent excisional biopsy of the breast lesion, and the 11 patients with pure DCIS at definitive histology, underwent SLN biopsy and, if required, definitive treatment of the primary tumour as a second operation.A total of 147 SLNs were identified and excised from the 102 patients: a single SLN was found in 61 cases (59.8%), two SLNs in 37 (36.3%), and three SLNs in 4 (3.9%).A positive SLN was found in one (0.98%) patient, who had one micrometastases (0.6 mm) detected by haematoxylin-eosin staining. The pathologic review of the surgical specimen confirmed that the tumour had been completely excised, and no microinfiltration was detected. In this SLN positive case, the primary tumour was a micropapillary DCIS, G2, with a diameter of 16 mm. The patient did not undergone previous microbiopsy or FNAC, and SLN biopsy was performed at the time of the primary tumour excision. Completion ALND was not performed.Discussion Indications for routine SLN biopsy in patients with a diagnosis of pure DCIS are still controversial. The first study proposing SLN biopsy for DCIS patients came from the H. Lee Moffitt Cancer Center in the year 2000 [ 7 ]: in a study of 87 DCIS patients, 5 (5.7%) presented SLN metastases, and the authors concluded that SLN biopsy should be routinely used in DCIS patients in order to identify and correctly stage patients with undetected invasive disease. In a later report on 195 DCIS patients from the same Institution, 26 (13%) patients were SLN positive and this finding strengthened the recommendation that SLN biopsy should become a routine part of surgical treatment for all DCIS patients [ 8 ]. The Memorial Sloan-Kettering Cancer Center group reviewed patients with "high risk" DCIS and found that 9/76 (12%) patients had SLNs positive for metastases. The authors suggested that SLN biopsy should be performed in all DCIS patients with one or more of the following characteristics: palpable or mammographic mass, suspicion of microinvasion, multicentric disease, high nuclear grade or necrosis [ 9 ]. On the other hand, in subsequent studies negligible rates of nodal involvement were found in patients with pure DCIS: Kelly et al. [ 10 ] found nodal metastases in only 3/134 (2.2%) patients, Intra et al. reported SLN metastases in 7/223 (3.1%) patients [ 11 ], Farkas et al. found no cases of SLN metastasis among 44 patients [ 12 ]. All these investigators concluded that SLN biopsy should not be routinely performed in patients with DCIS, with the exception of those undergoing mastectomy, because this operation precludes the possibility of performing a subsequent SLN biopsy if invasive foci are found at histology of the mastectomy specimen. Our results are consistent with the latter series of reports, since we found only one case of SLN micrometastasis among 102 patients with a diagnosis of pure DCIS (0.98%). The variability in the reported rates of nodal metastases probably reflects differences in the accuracy of pathological evaluation of the primary breast tumour: extensive sampling and a thorough histological examination of the DCIS are of crucial importance in ruling out microinvasive foci. Microinvasive DCIS is a different pathological entity, with a well-defined metastatic potential, and should be excluded from studies evaluating the role of axillary staging in non-invasive cancer. Our results support the view that the presence of axillary nodal metastases in patients with a final histopathological diagnosis of pure DCIS is a very unusual phenomenon, if the primary breast tumour has been completely excised and thoroughly examined by the pathologist. We, therefore, believe that routine SLN biopsy in all DCIS patients represents an over-treatment and should be avoided. Enthusiasm for SLN biopsy in DCIS patients is partly due to the low morbidity of the procedure. However, complications such as lymphedema, seroma, infection and sensory neuropathy have been reported after SLN biopsy [ 13 - 15 ]. Moreover, performing SLN biopsy in patients with a small DCIS treated with a conservative operation precludes the possibility of using this procedure in patients with a subsequent homolateral invasive tumour, which is not infrequent in this setting [ 1 ]. Finally, the policy of performing primary tumour excision with simultaneous SLN biopsy in all patients with a preoperative diagnosis of DCIS may incur a risk of performing axillary biopsy in patients with benign breast lesions, since intraoperative frozen section histology is usually unreliable in patients with small areas of microcalcifications. We therefore believe that this procedure should be used cautiously, being reserved for cases in which a real advantage can be expected. Another factor that has prompted interest in SLN biopsy for DCIS patients is the widespread use of image-guided core needle biopsy for the diagnosis of mammographically-detected abnormalities, a diagnostic technique that often fails to identify invasive foci: 14 to 29% of patients with a preoperative core needle biopsy of DCIS are found to have invasive cancer at surgical excision [ 16 , 17 ], and require a second operation for axillary staging. With vacuum-assisted large core biopsy the under-diagnosis rate is lower, but it is still not negligible. Supporters of routine SLN biopsy claim that this procedure is not reliable after primary tumour excision. They therefore believe that all patients with a preoperative diagnosis of DCIS who undergo definitive surgery and are upstaged because the pathologist finds foci of invasion on the specimen, must undergo an ALND if a SLN biopsy was not performed at the time of the first operation [ 18 ]. We disagree with this view, because it has been clearly demonstrated that SLN biopsy can be safely performed as a second procedure after primary tumour excision [ 19 , 20 ]. The only exceptions to this are patients who undergo mastectomy and those who require a wide quadrantectomy of the upper outer quadrant, which can disrupt lymphatic pathways toward the axilla. We therefore believe that only patients with a preoperative diagnosis of DCIS who need mastectomy or wide excision close to the axilla should undergo a concomitant SLN biopsy. In all other cases, only the primary tumour excision should be performed and SLN biopsy should be reserved, as a second procedure, for patients found to have infiltration at the histologic examination of the primary tumour. It has been claimed that some features of DCIS (large dimensions, high grade, comedo-type, mass forming lesions) are associated with a higher risk of invasive disease and nodal metastases. Therefore, some authors suggest that SLN biopsy should be reserved for all patients with these "high- risk" DCIS [ 9 , 21 , 22 ]. However, several investigators fail to correlate any histopathologic parameter with lymph node metastases [ 8 , 10 , 11 ]. The majority of patients in our series presented with low risk DCIS: most of the tumours were small (<1 cm) and only 20/102 patients had a palpable breast mass. Therefore, we cannot rule out that a higher incidence of microinvasion and nodal metastases might be found in a patient population with more aggressive forms of DCIS. However, in our series, the one DCIS patient with SLN metastasis had a small micropapillary, G2, non-palpable tumour. In our opinion, clinical judgement should be used in patients with large solid tumours or diffuse comedo-type DCIS, bearing in mind that most of these patients require a mastectomy and are therefore candidates for SLN biopsy.
Discussion
Indications for routine SLN biopsy in patients with a diagnosis of pure DCIS are still controversial. The first study proposing SLN biopsy for DCIS patients came from the H. Lee Moffitt Cancer Center in the year 2000 [ 7 ]: in a study of 87 DCIS patients, 5 (5.7%) presented SLN metastases, and the authors concluded that SLN biopsy should be routinely used in DCIS patients in order to identify and correctly stage patients with undetected invasive disease. In a later report on 195 DCIS patients from the same Institution, 26 (13%) patients were SLN positive and this finding strengthened the recommendation that SLN biopsy should become a routine part of surgical treatment for all DCIS patients [ 8 ].The Memorial Sloan-Kettering Cancer Center group reviewed patients with "high risk" DCIS and found that 9/76 (12%) patients had SLNs positive for metastases. The authors suggested that SLN biopsy should be performed in all DCIS patients with one or more of the following characteristics: palpable or mammographic mass, suspicion of microinvasion, multicentric disease, high nuclear grade or necrosis [ 9 ].On the other hand, in subsequent studies negligible rates of nodal involvement were found in patients with pure DCIS: Kelly et al. [ 10 ] found nodal metastases in only 3/134 (2.2%) patients, Intra et al. reported SLN metastases in 7/223 (3.1%) patients [ 11 ], Farkas et al. found no cases of SLN metastasis among 44 patients [ 12 ]. All these investigators concluded that SLN biopsy should not be routinely performed in patients with DCIS, with the exception of those undergoing mastectomy, because this operation precludes the possibility of performing a subsequent SLN biopsy if invasive foci are found at histology of the mastectomy specimen. Our results are consistent with the latter series of reports, since we found only one case of SLN micrometastasis among 102 patients with a diagnosis of pure DCIS (0.98%).The variability in the reported rates of nodal metastases probably reflects differences in the accuracy of pathological evaluation of the primary breast tumour: extensive sampling and a thorough histological examination of the DCIS are of crucial importance in ruling out microinvasive foci. Microinvasive DCIS is a different pathological entity, with a well-defined metastatic potential, and should be excluded from studies evaluating the role of axillary staging in non-invasive cancer.Our results support the view that the presence of axillary nodal metastases in patients with a final histopathological diagnosis of pure DCIS is a very unusual phenomenon, if the primary breast tumour has been completely excised and thoroughly examined by the pathologist. We, therefore, believe that routine SLN biopsy in all DCIS patients represents an over-treatment and should be avoided.Enthusiasm for SLN biopsy in DCIS patients is partly due to the low morbidity of the procedure. However, complications such as lymphedema, seroma, infection and sensory neuropathy have been reported after SLN biopsy [ 13 - 15 ]. Moreover, performing SLN biopsy in patients with a small DCIS treated with a conservative operation precludes the possibility of using this procedure in patients with a subsequent homolateral invasive tumour, which is not infrequent in this setting [ 1 ].Finally, the policy of performing primary tumour excision with simultaneous SLN biopsy in all patients with a preoperative diagnosis of DCIS may incur a risk of performing axillary biopsy in patients with benign breast lesions, since intraoperative frozen section histology is usually unreliable in patients with small areas of microcalcifications.We therefore believe that this procedure should be used cautiously, being reserved for cases in which a real advantage can be expected.Another factor that has prompted interest in SLN biopsy for DCIS patients is the widespread use of image-guided core needle biopsy for the diagnosis of mammographically-detected abnormalities, a diagnostic technique that often fails to identify invasive foci: 14 to 29% of patients with a preoperative core needle biopsy of DCIS are found to have invasive cancer at surgical excision [ 16 , 17 ], and require a second operation for axillary staging. With vacuum-assisted large core biopsy the under-diagnosis rate is lower, but it is still not negligible. Supporters of routine SLN biopsy claim that this procedure is not reliable after primary tumour excision. They therefore believe that all patients with a preoperative diagnosis of DCIS who undergo definitive surgery and are upstaged because the pathologist finds foci of invasion on the specimen, must undergo an ALND if a SLN biopsy was not performed at the time of the first operation [ 18 ]. We disagree with this view, because it has been clearly demonstrated that SLN biopsy can be safely performed as a second procedure after primary tumour excision [ 19 , 20 ]. The only exceptions to this are patients who undergo mastectomy and those who require a wide quadrantectomy of the upper outer quadrant, which can disrupt lymphatic pathways toward the axilla. We therefore believe that only patients with a preoperative diagnosis of DCIS who need mastectomy or wide excision close to the axilla should undergo a concomitant SLN biopsy. In all other cases, only the primary tumour excision should be performed and SLN biopsy should be reserved, as a second procedure, for patients found to have infiltration at the histologic examination of the primary tumour.It has been claimed that some features of DCIS (large dimensions, high grade, comedo-type, mass forming lesions) are associated with a higher risk of invasive disease and nodal metastases. Therefore, some authors suggest that SLN biopsy should be reserved for all patients with these "high- risk" DCIS [ 9 , 21 , 22 ]. However, several investigators fail to correlate any histopathologic parameter with lymph node metastases [ 8 , 10 , 11 ].The majority of patients in our series presented with low risk DCIS: most of the tumours were small (<1 cm) and only 20/102 patients had a palpable breast mass. Therefore, we cannot rule out that a higher incidence of microinvasion and nodal metastases might be found in a patient population with more aggressive forms of DCIS. However, in our series, the one DCIS patient with SLN metastasis had a small micropapillary, G2, non-palpable tumour.In our opinion, clinical judgement should be used in patients with large solid tumours or diffuse comedo-type DCIS, bearing in mind that most of these patients require a mastectomy and are therefore candidates for SLN biopsy.Conclusion Our results confirm that the finding of SLN metastasis in pure breast DCIS is a very rare occurence, if the primary tumour has been completely excised and microinvasion has been ruled out by a thorough histologic examination. Therefore, our current policy is to avoid routine SLN biopsy at the time of primary tumour resection in the presence of a preoperative diagnosis of DCIS. We reserve this procedure, as a second operation, for cases in which an invasive component is identified at the histologic examination of the surgical specimen. Only patients with a diagnosis of DCIS requiring a mastectomy or a wide resection close to the axilla should undergo concomitant SLN biopsy. However, particularly if a large high-grade tumour is found, patients should be informed of the risk that invasive disease may be found, and that a second procedure for SLN biopsy may be required. In these cases, the patient can decide whether to undergo a potentially unnecessary SLN biopsy at the time of the first operation or whether to run the risk of requiring a second operation.
Conclusion
Our results confirm that the finding of SLN metastasis in pure breast DCIS is a very rare occurence, if the primary tumour has been completely excised and microinvasion has been ruled out by a thorough histologic examination. Therefore, our current policy is to avoid routine SLN biopsy at the time of primary tumour resection in the presence of a preoperative diagnosis of DCIS. We reserve this procedure, as a second operation, for cases in which an invasive component is identified at the histologic examination of the surgical specimen. Only patients with a diagnosis of DCIS requiring a mastectomy or a wide resection close to the axilla should undergo concomitant SLN biopsy.However, particularly if a large high-grade tumour is found, patients should be informed of the risk that invasive disease may be found, and that a second procedure for SLN biopsy may be required. In these cases, the patient can decide whether to undergo a potentially unnecessary SLN biopsy at the time of the first operation or whether to run the risk of requiring a second operation.Competing interests The author(s) declare that they have no competing interests
Competing interests
The author(s) declare that they have no competing interestsAuthors' contributions GZ planned the study and drafted the manuscript PC, RM, ZF, GS, PB, PP, AB contributed with their own cases to the final multi-institutional series RM and GC reviewed the pathologic specimens of selected cases and drafted the histopatology section of the manuscript MEP and GM were in charge of data collection ML and DN co-ordinated the study
Authors' contributions
GZ planned the study and drafted the manuscriptPC, RM, ZF, GS, PB, PP, AB contributed with their own cases to the final multi-institutional seriesRM and GC reviewed the pathologic specimens of selected cases and drafted the histopatology section of the manuscriptMEP and GM were in charge of data collectionML and DN co-ordinated the studyPre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: