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Immobilization of antibodies and bacterial binding on nanodiamond and carbon nanotubes for biosensor applications
Diamond and Related Materials 13 (2004) 1098–1102
Immobilization of antibodies and bacterial binding on nanodiamond and carbon nanotubes for biosensor applications T.S. Huanga,e, Y. Tzengb,e,*, Y.K. Liuc,a, Y.C. Chenb,e, K.R. Walkera,e, R. Guntupallid,e, C. Liub,e a Department of Nutrition and Food Science, Auburn University, Auburn, AL 36849, USA Alabama Microelectronics Science and Technology Center, Department of Electrical and Computer Engineering, Auburn University, 200 Broun Hall, Auburn, AL 36849, USA c Power Mechanical Engineering, National Tsing-Hua University, Hsin-Chu, Taiwan, ROC d Department of Mechanical Engineering, Auburn University, Alabama 36849, USA e Peak of Excellence Center for Detection and Food Safety, Auburn University, Auburn, AL 36849, USA
b
Abstract High specific anti-Salmonella and anti-Staphylococcus aureus antibodies can be immobilized on hydrophobic and hydrophilic nanodiamond and carbon-nanotube coated silicon substrates. The efficacy of antibody immobilization was evaluated by enzyme linked immunosorbent assay (ELISA) and the bacterial binding efficiency was analyzed by SEM pictures. The immobilization efficacy of both antibodies and bacterial binding efficiency on air plasma treated nanodiamond are better than those of the hydrogen plasma treated. The results of antibody immobilization and S. aureus binding on the surfaces of hydrophobic and hydrophilic carbon nanotubes showed significant differences. The bacteria binding ability on hydrophilic carbon nanotubes is higher than hydrophobic carbon nanotubes. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Nanodiamond; Carbon nanotubes; Immobilization; Antibodies; Bacterial binding; Biosensor
1. Introduction Since the September 11 terrorist attack and the deaths from anthrax contamination, biosafety issues have become prominent as pathogens have the potential to become lethal weapons in the hands of terrorists. Food safety, especially with regard to foodborne pathogens, is another one of the most important issues today. Therefore, the rapid or real time detection of pathogens becomes extremely important for the food industry and government. Each year, as many as 76 million Americans become ill due to foodborne pathogens and toxins w1x. Foodborne illness can lead to as many as 1 million cases of permanent disabilities, including meningitis, gangrene, neurological effects and other chronic sequelae. The topsix illness-causing pathogens alone account for $6.9 billion in medical costs, productivity losses from missed work, and an estimated value of premature deaths w1x. The best way to reduce or prevent the incidences of *Corresponding author. Tel.: q1-334-844-1869; fax: q1-334-8441809. E-mail address: [email protected] (Y. Tzeng).
foodborne infection is by providing effective monitoring systems and good hygienic practices from food producers to consumers. The use of antibodies as probes for the detection of bacteria and other biological agents has been extensively explored w2x. The capture of specific bacteria by particular antibodies results in a mass change that can be detected by different sensor platforms. For example, the acoustic wave sensor, in which a piece of piezoelectric material is used to form a resonator w3–10x may offer a solution to meet the requirements for the effective detection of agents of interest. The cutting-edge technologies of nanodiamonds and carbon nanotubes have been investigated extensively and are potential candidates to be applied in the development of next generation biosensors. However, the immobilization of antibodies on the sensor platform to convert a non-electrical, physical or chemical, quantity into an electrical signal is the key for the control and the improvement of the performance of such a biosensor. Several immobilization methods have been reported for the improvement of the antibody–antigen binding to increase detection sensitivity
0925-9635/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.diamond.2003.11.047
T.S. Huang et al. / Diamond and Related Materials 13 (2004) 1098–1102
or for covalent binding of antibody or protein on solid surface w6,11–13x. In this study, the antibody immobilization on the surfaces of various oxidation processed nanodiamond and carbon nanotubes has been investigated.
1099
power at a pressure of 40 Torr and was used for the specimen exposure that lasted for a few minutes. Diamond was exposed to air plasmas generated at 1 kW microwave power for a few minutes to reduce the surface hydrogen termination and increase the termination with O and OH radicals.
2. Experimental details 2.1. Bacterial preparation Salmonella typhimurium and Staphylococcus aureus were grown in trypticase soy broth (TSB) and incubated at 37 8C in gyratory water bath at 100 rev.ymin for 15 h. The bacteria were washed twice with phosphate buffer saline (PBS) by performing centrifugation of the broth at 3500=g for 10 min and inactivated by re-suspending in 0.1% formalin PBS for 12 h at room temperature. The inactivated bacterial cells were washed twice with PBS. The bacterial numbers were estimated before inactivation by measuring the absorbance of bacterial suspension at 640 nm and calculating through the constructed standard curve and were confirmed by spread-plate method. The concentration of the bacterial suspension was adjusted to 108 cfuyml using PBS. 2.2. Antibodies preparation The polyclonal antibodies of anti-Salmonella and antiS. aureus rabbit immunoglobulins (IgGs) were produced following the protocols from Green and Manson w14x. The IgGs were purified from collected rabbit serum through multiple steps of ammonium sulfate precipitation and affinity Protein-A column chromatography w15x. The titer, IgG content, reactivity, and specificity of the purified IgG were tested using enzyme linked immunosorbent assay (ELISA) method w16x. 2.3. Nanodiamond preparation Nanocrystalline diamond films were deposited on silicon substrate in AsTex 1.5 kW 2.45 GHz microwave reactor from the pre-mixture gases 1%CH4q5%H2q 94%Ar. Before deposition, silicon substrates were dipped into buffered hydrofluoric acid etchant for 10 min to remove silicon dioxide and then were scratched by 1y4 mm diamond paste followed by immersing in nanodiamondymethanol solution in an ultrasonic bath for 1 h to improve the nucleation density. The as-grown nanocrystalline diamond films then were treated with air and hydrogen plasmas, respectively, to modify the surface properties for antibody immobilization. Both plasma oxidation and hydrogenation processes were carried out by exposing the surfaces of the diamond specimens to air plasma and hydrogen plasma, respectively, in a cylindrical microwave plasma reactor. The hydrogen plasma was generated at 1 kW of microwave
2.4. Carbon nanotube preparation Carbon nanotubes were grown by a thermal chemical vapor deposition process in a gas mixture of argon and acetylene. After sputtering iron catalyst thin films onto silicon substrates and oxidizing to form nanoparticles, the carbon-nanotube growth process was carried out in a vacuum furnace heated to 700 8C, and the growth was done at a pressure of 75 Torr for 20 min. The length of vertically aligned carbon nanotubes was approximately 20 mm. The hydrophilic carbon nanotubes were obtained by placing the carbon-nanotube coated silicon substrates on a hot plate at 400 8C in air for 20 min to oxidize the tips of carbon nanotubes. Testing of thermally oxidized carbon nanotubes by means of a water drop resulted in similar hydrophilic behavior. 2.5. Antibody immobilization and ELISA test The test was performed at room temperature. Rabbit IgGs were coated on the treated and untreated nanodiamonds and carbon nanotubes for 2 h. After three washings with washing buffer (0.1% Tween20, 0.02% NaN3 PBS), the unbound areas of samples were blocked with 3% BSA for 1 h. Then, the 108 cfuyml of S. typhimurium and S. aureus suspensions were added to the antibody coated plates, respectively, for 1 h. Following three washings, the rabbit anti-S. typhimurium IgG alkaline phosphatase conjugate were added to the plates and incubated for 1 h. The enzyme substrate of pnitrophenyl phosphate was added and incubated for 30 min. After stopping the reaction by adding 2 M NaOH, the absorbance at 405 nm was recorded for the evaluation of antibody binding efficiency. 2.6. Antibody and bacteria binding The antibodies were immobilized on the surfaces of prepared samples for 2 h. After coating, the samples were washed three times with washing buffer and then 108 cellsyml of S. typhimurium and S. aureus suspensions were added and incubated for 1 h. Then, the samples were washed three times with deionized water to remove unbound bacteria and air dried for 3 min. The bound bacteria were fixed with osmium tetroxide gas for 5 h, and then coated with a thin layer of gold for SEM observation. The results were recorded photographically.
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Fig. 1. The specificity of anti-Salmonella and anti-S. aureus rabbit IgGs with various bacteria using ELISA. A: S. typhimurium; B: S. mission; C: Salmonella paratyphi; D: Salmonella montevedo; E: S. enteritidis; F: Escherichia coli O157:H7; G: L. monocytogenes; H: S. aureus.
3. Results and discussion Both of the produced anti-Salmonella and anti-S. aureus rabbit IgGs have high specificity. The antiSalmonella antibody can react with S. typhimurium, Salmonella mission, and Salmonella enteritidis and slightly react with S. aureus. The anti-S. aureus antibody is able to react with S. aureus and slightly react with Listeria monocytogenes (Fig. 1). The titers for these two antibodies are over 20 000 times dilution. The anti-S. aureus and anti-Salmonella rabbit IgGs can be immobilized on the surfaces of nanodiamonds treated with air and hydrogen plasma. There is no significant difference of immobilization among these two antibodies on both the air and hydrogen plasma treated samples. The immobilization efficacy of antibody on air plasma treated nanodiamond is better than that of the hydrogen plasma treated (Fig. 2). The surface hydrophobicity of nanodiamond may be reduced by the treatment of air plasma resulting in an increase in the hydrophilicity to facilitate the binding of antibodies. Although the anti-S. aureus and anti-Salmonella rabbit IgGs can be immobilized on the aligned hydrophobic and hydrophilic carbon nanotubes, the immobilization efficacy on hydrophilic carbon nanotubes is better than on hydrophobic carbon nanotubes and has been shown significant difference. There are similar immobilization effectiveness of both antibodies on hydrophobic and hydrophilic CNTs (Fig. 3). The S. aureus can be bound to the antibody immobilized nanodiamond. The bacteria binding efficiency on hydrophilic nanodiamond is better than on hydrophobic nanodiamond (Fig. 4). These results agree with the data from the immobilization efficacy assay by ELISA. The treatment of air plasma may cause a surface change on the nanodiamond by increasing the hydrogen bond between antibody and nanodiamond. However, it needs
Fig. 2. The immobilization efficacy of anti-Salmonella and anti-S. aureus rabbit IgGs on air plasma and hydrogen plasma treated nanodiamonds using ELISA.
to be confirmed with further testing. The S. typhimurium can also be bound to the surface of antibody immobilized nanodiamond (Fig. 5). These results confirmed the data assayed by ELISA in antibody immobilization in which the air plasma treated nanodiamond has higher antibody immobilization efficacy. There is more than 50% surface area of hydrophilic nanodiamod covered by this bacterium. The binding efficiency of S. typhimurium is higher than S. aureus as shown in Figs. 4 and 5. The antibody immobilization and S. aureus binding on the surfaces of hydrophobic and hydrophilic carbon nanotubes also has been performed. The result showed that the bacteria binding ability on hydrophilic carbon nanotubes is higher than hydrophobic carbon nanotubes (Fig. 6). Both data from ELISA assay and SEM pictures have proven that the heat treated carbon nanotubes are able to increase the ability of antibody immobilization.
Fig. 3. The immobilization efficacy of anti-Salmonella and anti-S. aureus rabbit IgGs on hydrophobic and hydrophilic carbon nanotubes using ELISA.
T.S. Huang et al. / Diamond and Related Materials 13 (2004) 1098–1102
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Fig. 4. The antibody immobilization and S. aureus binding on the surfaces of nanodiamond. (a) Control (air plasma treated without antibody immobilization); (b) hydrogen plasma treated; (c) air plasma treated.
Fig. 5. The antibody immobilization and S. typhimurium binding on the surfaces of nanodiamond. (a) Control (air plasma treated without antibody immobilization); (b) hydrogen plasma treated; (c): air plasma treated.
Fig. 6. The antibody immobilization and S. aureus binding on the surfaces of carbon nanotubes. (a) Control (hydrophilic CNT without antibody immobilization); (b) hydrophobic CNT; (c) hydrophilic CNT.
The heat treatment in air caused the oxidization of the carbon nanotubes. The physical adsorption of antibody immobilization on solid phase is dependent on the non-specific interaction between antibody and solid phase. These interactions include various non-covalent bonds, such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. Among these interaction forces, the hydrogen bond is the strongest.
The number and extent of these interactions mainly depend on the chemical properties of antibodies and the solid surfaces as well as the solvent w17,18x. The surfaces of diamond that were exposed to hydrogen plasmas were terminated with atomic hydrogen and were hydrophobic. The surfaces of as-grown carbon nanotubes are hydrophobic, too. According to the chemical and physical properties, the hydrophilic solution is difficult to distribute on the hydrophobic surface evenly.
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T.S. Huang et al. / Diamond and Related Materials 13 (2004) 1098–1102
The oxidized surfaces of nanodiamond and carbon nanotubes became more hydrophilic and were terminated with O or OH to form C_ O and C–OH. The contact between the hydrophilic antibody solution and the surfaces of hydrophilic nanodiamond and carbon nanotubes was, therefore, enhanced. The interaction forces between antibodies and surfaces were also promoted, and the quantity of antibody immobilization increased. The immobilized antibodies on the surfaces of diamond and carbon nanotubes were sufficiently stable for the successful binding of bacteria as demonstrated in this work. More details of the interaction forces that are involved in the success of the immobilization of antibodies and the successful binding of various bacteria on diamond surfaces and carbon nanotube surfaces are being further investigated. 4. Conclusion Both anti-Salmonella and anti-S. aureus rabbit antibodies can be immobilized on hydrophobic and hydrophilic nanodiamond and carbon nanotube coated silicon substrates. The air plasma treated nanodiamond has a higher efficacy of antibody immobilization and ability of bacterial binding than hydrogen plasma treated nanodiamond. The air plasma oxidization process at 400 8C for 20 min is able to increase the hydrophilic property of aligned carbon nanotube. This resulted in promoting the immobilization of anti-S. aureus rabbit IgG and bacterial binding. References w1x P.S. Mead, L. Slutsker, V. Dietz, L.F. McCaig, J.S. Bresee, C. Shapiro, et al., Food-related illness and death in the United States, Emerging Infect. Dis. 5 (1999) 607. w2x S.S. Iqbal, M.W. Mayo, J.G. Bruno, B.V. Bronk, C.A. Batt, J.P. Chambers, A review of molecular recognition technologies for detection of biological threat agents, Biosens. Bioelectron. 15 (2000) 549. w3x S. Babacan, P. Pivarnik, S. Letcher, A.G. Rand, Evaluation of antibody immobilization methods for piezoelectric biosensor applications, Biosens. Bioelectron. 15 (2000) 615.
w4x F. Bender, R.W. Cernosek, F. Josse, Love-wave biosensor using cross-linked polymer waveguides on LiTaO3 substrates, Electron. Lett. 36 (2000) 1672. w5x R. Lucklum, P. Haauptmann, The quartz crystal microbalance: mass sensitivity, viscoelasticity and acoustic amplification, Sensor Actuat. B 70 (2000) 30. w6x I.S. Park, N. Kim, Thiolated Salmonella antibody immobilization onto the gold surface of piezoelectric quartz crystal, Biosens. Bioelectron. 13 (1998) 1091. w7x S.T. Pathirana, J. Barbaree, B.A. Chin, M.G. Hartell, W.C. Neely, V. Vodyanoy, Rapid and sensitive biosensor for salmonella, Biosens. Bioelectron. 15 (2000) 135. w8x J. Rickert, A. Brecht, W. Gopel, Quartz crystal microbalance for quantitative biosensing characterizing protein multilayers, Biosens. Bioelectron. 12 (1997) 567. w9x X.D. Su, S. Low, J. Kwang, V.H.T. Chew, S.F.Y. Li, Piezoelectric quartz crystal based veterinary diagnosis for Salmonella enteritidis infection in chicken and egg, Sensor Actuat. B 75 (2001) 29. w10x Y.Y. Wong, S.P. Ng, M.H. Ng, S.H. Si, S.Z. Yao, Y.S. Fung, Immunosensor for the differentiation and detection of salmonella species based on a quartz crystal microbalance, Biosens. Bioelectron. 17 (2002) 676. w11x Z. Bilkova, J. Mazurova, J. Churacek, D. Horak, J. Turkova, Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a non-porous solid support, J. Chromatogr. A 852, 41. w12x S. Busse, V. Scheumann, B. Menges, S. Mittler, Sensitivity studies for specific binding reactions sing the biotinystreptavidin system by evanescent optical methods, Biosens. Bioelectron. 17 (2002) 704. w13x O. Ouerghi, A. Touhami, N. Jaffrezic-Renault, C. Martelet, H. Ben Ouada, S. Cosnier, Impedimetric immunosensor using avidin-biotin for antibody immobilization, Bioelectrochemistry 56 (2002) 131. w14x J.A. Green, M.M. Manson, Production of polyclonal antisera, in: M.M. Manson (Ed.), Immunochemical Protocols, Humana Press, Totowa, New Jersey, 1992, pp. 1–5. w15x E. Harlow, D. Lane, Chapter 8: storing and purifying antibodies, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Express, Cold Spring Harbor, New York, 1988, p. 283. w16x T.S. Huang, M.R. Marshall, K.J. Kao, W.S. Otwell, C.I. Wei, Development of monoclonal antibody for red snapper (Lutjanus campechanus) identification using enzyme-linked immunosorbent assay, J. Agric. Food Chem. 43 (1995) 2301. w17x L.A. Cantarero, J.E. Butler, J.W. Osborne, The adsorptive characteristics of proteins for polystyrene and their significance in sold-phase immunoassays, Anal. Biochem. 105 (1980) 375. w18x M. Nisnevitch, M.A. Firer, The solid phase in affinity chromatography: strategies for antibody attachment, J. Biochem. Biophys. Method 49 (2001) 467.
Immobilization of antibodies and bacterial binding on nanodiamond and carbon nanotubes for biosensor applications T.S. Huanga,e, Y. Tzengb,e,*, Y.K. Liuc,a, Y.C. Chenb,e, K.R. Walkera,e, R. Guntupallid,e, C. Liub,e a Department of Nutrition and Food Science, Auburn University, Auburn, AL 36849, USA Alabama Microelectronics Science and Technology Center, Department of Electrical and Computer Engineering, Auburn University, 200 Broun Hall, Auburn, AL 36849, USA c Power Mechanical Engineering, National Tsing-Hua University, Hsin-Chu, Taiwan, ROC d Department of Mechanical Engineering, Auburn University, Alabama 36849, USA e Peak of Excellence Center for Detection and Food Safety, Auburn University, Auburn, AL 36849, USA
b
Abstract High specific anti-Salmonella and anti-Staphylococcus aureus antibodies can be immobilized on hydrophobic and hydrophilic nanodiamond and carbon-nanotube coated silicon substrates. The efficacy of antibody immobilization was evaluated by enzyme linked immunosorbent assay (ELISA) and the bacterial binding efficiency was analyzed by SEM pictures. The immobilization efficacy of both antibodies and bacterial binding efficiency on air plasma treated nanodiamond are better than those of the hydrogen plasma treated. The results of antibody immobilization and S. aureus binding on the surfaces of hydrophobic and hydrophilic carbon nanotubes showed significant differences. The bacteria binding ability on hydrophilic carbon nanotubes is higher than hydrophobic carbon nanotubes. 䊚 2003 Elsevier B.V. All rights reserved. Keywords: Nanodiamond; Carbon nanotubes; Immobilization; Antibodies; Bacterial binding; Biosensor
1. Introduction Since the September 11 terrorist attack and the deaths from anthrax contamination, biosafety issues have become prominent as pathogens have the potential to become lethal weapons in the hands of terrorists. Food safety, especially with regard to foodborne pathogens, is another one of the most important issues today. Therefore, the rapid or real time detection of pathogens becomes extremely important for the food industry and government. Each year, as many as 76 million Americans become ill due to foodborne pathogens and toxins w1x. Foodborne illness can lead to as many as 1 million cases of permanent disabilities, including meningitis, gangrene, neurological effects and other chronic sequelae. The topsix illness-causing pathogens alone account for $6.9 billion in medical costs, productivity losses from missed work, and an estimated value of premature deaths w1x. The best way to reduce or prevent the incidences of *Corresponding author. Tel.: q1-334-844-1869; fax: q1-334-8441809. E-mail address: [email protected] (Y. Tzeng).
foodborne infection is by providing effective monitoring systems and good hygienic practices from food producers to consumers. The use of antibodies as probes for the detection of bacteria and other biological agents has been extensively explored w2x. The capture of specific bacteria by particular antibodies results in a mass change that can be detected by different sensor platforms. For example, the acoustic wave sensor, in which a piece of piezoelectric material is used to form a resonator w3–10x may offer a solution to meet the requirements for the effective detection of agents of interest. The cutting-edge technologies of nanodiamonds and carbon nanotubes have been investigated extensively and are potential candidates to be applied in the development of next generation biosensors. However, the immobilization of antibodies on the sensor platform to convert a non-electrical, physical or chemical, quantity into an electrical signal is the key for the control and the improvement of the performance of such a biosensor. Several immobilization methods have been reported for the improvement of the antibody–antigen binding to increase detection sensitivity
0925-9635/04/$ - see front matter 䊚 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.diamond.2003.11.047
T.S. Huang et al. / Diamond and Related Materials 13 (2004) 1098–1102
or for covalent binding of antibody or protein on solid surface w6,11–13x. In this study, the antibody immobilization on the surfaces of various oxidation processed nanodiamond and carbon nanotubes has been investigated.
1099
power at a pressure of 40 Torr and was used for the specimen exposure that lasted for a few minutes. Diamond was exposed to air plasmas generated at 1 kW microwave power for a few minutes to reduce the surface hydrogen termination and increase the termination with O and OH radicals.
2. Experimental details 2.1. Bacterial preparation Salmonella typhimurium and Staphylococcus aureus were grown in trypticase soy broth (TSB) and incubated at 37 8C in gyratory water bath at 100 rev.ymin for 15 h. The bacteria were washed twice with phosphate buffer saline (PBS) by performing centrifugation of the broth at 3500=g for 10 min and inactivated by re-suspending in 0.1% formalin PBS for 12 h at room temperature. The inactivated bacterial cells were washed twice with PBS. The bacterial numbers were estimated before inactivation by measuring the absorbance of bacterial suspension at 640 nm and calculating through the constructed standard curve and were confirmed by spread-plate method. The concentration of the bacterial suspension was adjusted to 108 cfuyml using PBS. 2.2. Antibodies preparation The polyclonal antibodies of anti-Salmonella and antiS. aureus rabbit immunoglobulins (IgGs) were produced following the protocols from Green and Manson w14x. The IgGs were purified from collected rabbit serum through multiple steps of ammonium sulfate precipitation and affinity Protein-A column chromatography w15x. The titer, IgG content, reactivity, and specificity of the purified IgG were tested using enzyme linked immunosorbent assay (ELISA) method w16x. 2.3. Nanodiamond preparation Nanocrystalline diamond films were deposited on silicon substrate in AsTex 1.5 kW 2.45 GHz microwave reactor from the pre-mixture gases 1%CH4q5%H2q 94%Ar. Before deposition, silicon substrates were dipped into buffered hydrofluoric acid etchant for 10 min to remove silicon dioxide and then were scratched by 1y4 mm diamond paste followed by immersing in nanodiamondymethanol solution in an ultrasonic bath for 1 h to improve the nucleation density. The as-grown nanocrystalline diamond films then were treated with air and hydrogen plasmas, respectively, to modify the surface properties for antibody immobilization. Both plasma oxidation and hydrogenation processes were carried out by exposing the surfaces of the diamond specimens to air plasma and hydrogen plasma, respectively, in a cylindrical microwave plasma reactor. The hydrogen plasma was generated at 1 kW of microwave
2.4. Carbon nanotube preparation Carbon nanotubes were grown by a thermal chemical vapor deposition process in a gas mixture of argon and acetylene. After sputtering iron catalyst thin films onto silicon substrates and oxidizing to form nanoparticles, the carbon-nanotube growth process was carried out in a vacuum furnace heated to 700 8C, and the growth was done at a pressure of 75 Torr for 20 min. The length of vertically aligned carbon nanotubes was approximately 20 mm. The hydrophilic carbon nanotubes were obtained by placing the carbon-nanotube coated silicon substrates on a hot plate at 400 8C in air for 20 min to oxidize the tips of carbon nanotubes. Testing of thermally oxidized carbon nanotubes by means of a water drop resulted in similar hydrophilic behavior. 2.5. Antibody immobilization and ELISA test The test was performed at room temperature. Rabbit IgGs were coated on the treated and untreated nanodiamonds and carbon nanotubes for 2 h. After three washings with washing buffer (0.1% Tween20, 0.02% NaN3 PBS), the unbound areas of samples were blocked with 3% BSA for 1 h. Then, the 108 cfuyml of S. typhimurium and S. aureus suspensions were added to the antibody coated plates, respectively, for 1 h. Following three washings, the rabbit anti-S. typhimurium IgG alkaline phosphatase conjugate were added to the plates and incubated for 1 h. The enzyme substrate of pnitrophenyl phosphate was added and incubated for 30 min. After stopping the reaction by adding 2 M NaOH, the absorbance at 405 nm was recorded for the evaluation of antibody binding efficiency. 2.6. Antibody and bacteria binding The antibodies were immobilized on the surfaces of prepared samples for 2 h. After coating, the samples were washed three times with washing buffer and then 108 cellsyml of S. typhimurium and S. aureus suspensions were added and incubated for 1 h. Then, the samples were washed three times with deionized water to remove unbound bacteria and air dried for 3 min. The bound bacteria were fixed with osmium tetroxide gas for 5 h, and then coated with a thin layer of gold for SEM observation. The results were recorded photographically.
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Fig. 1. The specificity of anti-Salmonella and anti-S. aureus rabbit IgGs with various bacteria using ELISA. A: S. typhimurium; B: S. mission; C: Salmonella paratyphi; D: Salmonella montevedo; E: S. enteritidis; F: Escherichia coli O157:H7; G: L. monocytogenes; H: S. aureus.
3. Results and discussion Both of the produced anti-Salmonella and anti-S. aureus rabbit IgGs have high specificity. The antiSalmonella antibody can react with S. typhimurium, Salmonella mission, and Salmonella enteritidis and slightly react with S. aureus. The anti-S. aureus antibody is able to react with S. aureus and slightly react with Listeria monocytogenes (Fig. 1). The titers for these two antibodies are over 20 000 times dilution. The anti-S. aureus and anti-Salmonella rabbit IgGs can be immobilized on the surfaces of nanodiamonds treated with air and hydrogen plasma. There is no significant difference of immobilization among these two antibodies on both the air and hydrogen plasma treated samples. The immobilization efficacy of antibody on air plasma treated nanodiamond is better than that of the hydrogen plasma treated (Fig. 2). The surface hydrophobicity of nanodiamond may be reduced by the treatment of air plasma resulting in an increase in the hydrophilicity to facilitate the binding of antibodies. Although the anti-S. aureus and anti-Salmonella rabbit IgGs can be immobilized on the aligned hydrophobic and hydrophilic carbon nanotubes, the immobilization efficacy on hydrophilic carbon nanotubes is better than on hydrophobic carbon nanotubes and has been shown significant difference. There are similar immobilization effectiveness of both antibodies on hydrophobic and hydrophilic CNTs (Fig. 3). The S. aureus can be bound to the antibody immobilized nanodiamond. The bacteria binding efficiency on hydrophilic nanodiamond is better than on hydrophobic nanodiamond (Fig. 4). These results agree with the data from the immobilization efficacy assay by ELISA. The treatment of air plasma may cause a surface change on the nanodiamond by increasing the hydrogen bond between antibody and nanodiamond. However, it needs
Fig. 2. The immobilization efficacy of anti-Salmonella and anti-S. aureus rabbit IgGs on air plasma and hydrogen plasma treated nanodiamonds using ELISA.
to be confirmed with further testing. The S. typhimurium can also be bound to the surface of antibody immobilized nanodiamond (Fig. 5). These results confirmed the data assayed by ELISA in antibody immobilization in which the air plasma treated nanodiamond has higher antibody immobilization efficacy. There is more than 50% surface area of hydrophilic nanodiamod covered by this bacterium. The binding efficiency of S. typhimurium is higher than S. aureus as shown in Figs. 4 and 5. The antibody immobilization and S. aureus binding on the surfaces of hydrophobic and hydrophilic carbon nanotubes also has been performed. The result showed that the bacteria binding ability on hydrophilic carbon nanotubes is higher than hydrophobic carbon nanotubes (Fig. 6). Both data from ELISA assay and SEM pictures have proven that the heat treated carbon nanotubes are able to increase the ability of antibody immobilization.
Fig. 3. The immobilization efficacy of anti-Salmonella and anti-S. aureus rabbit IgGs on hydrophobic and hydrophilic carbon nanotubes using ELISA.
T.S. Huang et al. / Diamond and Related Materials 13 (2004) 1098–1102
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Fig. 4. The antibody immobilization and S. aureus binding on the surfaces of nanodiamond. (a) Control (air plasma treated without antibody immobilization); (b) hydrogen plasma treated; (c) air plasma treated.
Fig. 5. The antibody immobilization and S. typhimurium binding on the surfaces of nanodiamond. (a) Control (air plasma treated without antibody immobilization); (b) hydrogen plasma treated; (c): air plasma treated.
Fig. 6. The antibody immobilization and S. aureus binding on the surfaces of carbon nanotubes. (a) Control (hydrophilic CNT without antibody immobilization); (b) hydrophobic CNT; (c) hydrophilic CNT.
The heat treatment in air caused the oxidization of the carbon nanotubes. The physical adsorption of antibody immobilization on solid phase is dependent on the non-specific interaction between antibody and solid phase. These interactions include various non-covalent bonds, such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces. Among these interaction forces, the hydrogen bond is the strongest.
The number and extent of these interactions mainly depend on the chemical properties of antibodies and the solid surfaces as well as the solvent w17,18x. The surfaces of diamond that were exposed to hydrogen plasmas were terminated with atomic hydrogen and were hydrophobic. The surfaces of as-grown carbon nanotubes are hydrophobic, too. According to the chemical and physical properties, the hydrophilic solution is difficult to distribute on the hydrophobic surface evenly.
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T.S. Huang et al. / Diamond and Related Materials 13 (2004) 1098–1102
The oxidized surfaces of nanodiamond and carbon nanotubes became more hydrophilic and were terminated with O or OH to form C_ O and C–OH. The contact between the hydrophilic antibody solution and the surfaces of hydrophilic nanodiamond and carbon nanotubes was, therefore, enhanced. The interaction forces between antibodies and surfaces were also promoted, and the quantity of antibody immobilization increased. The immobilized antibodies on the surfaces of diamond and carbon nanotubes were sufficiently stable for the successful binding of bacteria as demonstrated in this work. More details of the interaction forces that are involved in the success of the immobilization of antibodies and the successful binding of various bacteria on diamond surfaces and carbon nanotube surfaces are being further investigated. 4. Conclusion Both anti-Salmonella and anti-S. aureus rabbit antibodies can be immobilized on hydrophobic and hydrophilic nanodiamond and carbon nanotube coated silicon substrates. The air plasma treated nanodiamond has a higher efficacy of antibody immobilization and ability of bacterial binding than hydrogen plasma treated nanodiamond. The air plasma oxidization process at 400 8C for 20 min is able to increase the hydrophilic property of aligned carbon nanotube. This resulted in promoting the immobilization of anti-S. aureus rabbit IgG and bacterial binding. References w1x P.S. Mead, L. Slutsker, V. Dietz, L.F. McCaig, J.S. Bresee, C. Shapiro, et al., Food-related illness and death in the United States, Emerging Infect. Dis. 5 (1999) 607. w2x S.S. Iqbal, M.W. Mayo, J.G. Bruno, B.V. Bronk, C.A. Batt, J.P. Chambers, A review of molecular recognition technologies for detection of biological threat agents, Biosens. Bioelectron. 15 (2000) 549. w3x S. Babacan, P. Pivarnik, S. Letcher, A.G. Rand, Evaluation of antibody immobilization methods for piezoelectric biosensor applications, Biosens. Bioelectron. 15 (2000) 615.
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