Editing Cabon Nanotube

<br>Quantitative lateral flow immunosensor using carbon nanotubes as label<br>Adeyabeba Abera and Jin-Woo Choi*<br>Received 28th June 2010, Accepted 26th August 2010<br>DOI: 10.1039/c0ay00412j<br>A novel immunosensor utilizing multi-walled carbon nanotube as label on a lateral flow system for<br>simple and quantitative electrical detection was investigated. Multi-walled carbon nanotubes<br>(MWCNTs) were first modified with polyvinylpyrrolidone (PVP) for uniform dispersion in aqueous<br>solution. Then, the MWCNTs were conjugated with human immunoglobulin G (IgG) using 1-(3-<br>(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC) coupling chemistry. The lateral<br>flow immunosensor was made of nitrocellulose membrane that transports sample and reagents through<br>a porous structure by capillary action. The performance of the immunosensor was demonstrated using<br>human IgG as a model analyte competing with the conjugate (MWCNT-labeled human IgG) at the<br>capture zone. As a result of binding reaction between the conjugate and the immobilized Protein A at<br>the capture zone, the conjugated MWCNTs formed a conducting network at the capture zone<br>providing conductance measurement corresponding to the amount of captured conjugate. Quantitative<br>immunoassay response for the target human IgG was demonstrated in the range of 25 to 200 μg mlÀ1<br>without an additional amplification step. The presented immunosensing technique could be expanded<br>for the detection of various analyte-specific biomolecules with a potential for simple and rapid tests<br>suitable for point-of-care diagnostics.<br>1. Introduction<br>Lateral flow (LF) immunoassay is one of the most successful<br>point-of-care diagnostic tools commercially available for detecting<br>various health and environmental agents.1–6<br>A lateral flow<br>immunosensor is made of a porous membrane for both immobi-<br>lizing biological receptors and transporting sample reagents.<br>Sample reagents are transported in the lateral direction through<br>the porous membrane by capillary force and captured by the<br>biological receptors immobilized at the capture zone. LF immu-<br>nosensors are simple to use, low-cost, rapid, and portable with<br>relatively long shelf life. Examples of lateral flow diagnostics<br>include home pregnancy tests, detection of drugs of abuse, diag-<br>nosis of infectious diseases, and cancer markers. However, such<br>systems often suffer from low sensitivity, and are mostly restricted<br>to qualitative or semi-quantitative measurements.<br>Generally, LF immunosensors are based on optical detection<br>where tagged particles or enzymes provide visible signal generation<br>or amplification. Typically, colloidal gold is used as a label for<br>colorimetric detection traced by the visible color. Several efforts<br>have been made towards signal quantification using enhanced<br>colorimetric labels or conducting materials such as phosphorescent<br>nanoparticles,7 polyaniline,8 and polyaniline with colloidal gold.9<br>Colorimetric techniques are often limited by low sensitivity while<br>fluorescent techniques suffer from instability, photobleaching,<br>activation step, and requirement for a complicated reader device.<br>This work addresses such limitations by utilizing carbon<br>nanotubes (CNTs) as labeling material for direct electrical signal<br>measurement. CNTs have remarkable one-dimensional elec-<br>tronic conduction properties and large surface area, and allow<br>the adsorption of biomolecules for sensing.10–12 In order to utilize<br>these properties of carbon nanotubes for biosensing applications,<br>surface functionalization is necessary to uniformly disperse<br>CNTs in aqueous solution and immobilize biorecognition<br>molecules on the surface of the nanotubes. Adsorption of<br>surfactants and polymers on the surface of CNTs is considered as<br>an effective method to enhance the dispersion of CNTs without<br>affecting their properties.13–15 Compared to CNTs, other con-<br>ducting materials such as colloidal gold and polymer nanowires<br>do not possess the unique material properties suitable for surface<br>functionalization or maintain high electrical conduction after<br>binding reaction.9,16<br>In this work, a new immunoassay technique using carbon<br>nanotube labels on lateral flow system is introduced. The illus-<br>tration of this technique is shown in Fig. 1. MWCNTs are<br>dispersed in water with PVP using sonication. Adsorption of<br>PVP provides a thin and rough layer of organic coating by<br>wrapping around the MWCNTs. Human IgG is immobilized on<br>the bare (not covered by PVP) surface of the MWCNTs. The<br>MWCNT–human IgG conjugate is then applied on a lateral flow<br>immunosensor where the MWCNT–human IgG conjugate is to<br>be captured by immobilized Protein A at the capture zone. The<br>conjugated MWCNTs form a conducting network at the capture<br>zone corresponding to the amount of binding. Consequently,<br>quantitative signal can be obtained by simple resistance<br>measurement of the capture zone. Blocking schemes were also<br>implemented on the MWCNTs and the lateral flow strips to<br>address non-specific interaction.<br>2. Methods and experiments<br>2.1 Materials and reagents<br>Carboxylated multi-walled carbon nanotubes (purity &gt; 95 wt%)<br>were obtained from Cheaptubes, Inc. (Brattleboro, VT). Hi-flow<br>Department of Electrical and Computer Engineering, Louisiana State<br>University, Baton Rouge, LA, 70803, USA; Web: www.ece.lsu.<br>edu/biomems/. E-mail: choi@ece.lsu.edu; Tel: +1-225-578-8764<br>This journal is © The Royal Society of Chemistry 2010<br>Anal. Methods, 2010, 2, 1819–1822 | 1819<br>TECHNICAL NOTE<br>www.rsc.org/methods | Analytical Methods<br>Downloaded on 30 December 2012<br>Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J<br>View Article Online / Journal Homepage / Table of Contents for this issue<br>Page 2<br>Plus nitrocellulose membranes (HF13502), glass fiber pads,<br>cellulose membranes, and a supporting card for lateral flow assay<br>were obtained from Millipore (Bedford, MA). Protein A sus-<br>pended in solution was obtained from BioVision, Inc. (Mountain<br>View, CA). Purified human immunoglobulin G (IgG, 6.2 mg mlÀ1)<br>suspended in 0.01 M sodium phosphate (0.15 M NaCl, pH 7.4)<br>was obtained from Sigma-Aldrich (St Louis, MO). Poly-<br>vinylpyrrolidone (PVP, Mw 10 000), bovine serum albumin (BSA),<br>Tween-20, glutaraldehyde, and 2-(N-morpholino)ethanesulfonic<br>acid (MES) were from Sigma-Aldrich. 1-(3-(Dimethylamino)-<br>propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-<br>hydroxysulfosuccinimide (sulfo-NHS) were obtained from<br>Thermo Fisher Scientific, Inc. (Rockford, IL). All immunore-<br>agents were diluted in pH 7.4 phosphate saline buffer (PBS) unless<br>noted otherwise.<br>2.2 MWCNT–antibody conjugation<br>In order to start the conjugation process, 1.5 mg of carboxylated<br>MWCNTs were dispersed in 2 ml of deionized (DI) water with 0.6<br>mg of PVP. The solution was sonicated for 4 hours in an ultra-<br>sonic bath at 27 C to exfoliate the MWCNTs and accelerate the<br>dispersion. The dispersed solution was mixed with 1 ml of EDC<br>(0.4 M) and sulfo-NHS (0.1 M) in pH 6.1 MES buffer. The<br>solution was left to react for 30 minutes with gentle agitation at<br>room temperature. The mixture was microcentrifuged at 13 000<br>rpm for 3 minutes with PBS buffer and the supernatant was dis-<br>carded. The washing and re-suspending process was repeated<br>three times to remove excess reagents. Human IgG solution was<br>prepared at 200 μg mlÀ1 by diluting with PBS buffer for conju-<br>gation. The solution containing functionalized MWCNTs was<br>then mixed with 100 μl of the human IgG solution and left to react<br>overnight at 4 C. This mixture was centrifuged at 13 000 rpm for<br>3 minutes and the supernatant was discarded. The washing and re-<br>suspending process was repeated three times to remove unbound<br>human IgG, and the resulting MWCNT–antibody conjugate<br>solution was kept at 4 C until use.<br>2.3 Preparation of lateral flow immunosensors<br>A nitrocellulose membrane was used as a detection pad with<br>designated capture zone for immobilizing Protein A. Fiber glass<br>and cellulose membranes were used for sample application and<br>for absorption of excess reagents by placing them at the two ends<br>of the nitrocellulose membrane, respectively. The LF immuno-<br>strips were prepared following general procedure reported else-<br>where.7,9 First, a nitrocellulose membrane was saturated with<br>10% (v/v) methanol in deionized (DI) water for 30 minutes to<br>remove any residue and dried in air at room temperature. The<br>membrane was then treated with 0.5% (v/v) glutaraldehyde<br>solution to strengthen the immobilization of Protein A on the<br>nitrocellulose membrane and rinsed with DI water. After drying,<br>Protein A diluted in PBS buffer to 1 mg mlÀ1 was immobilized at<br>a designated capture zone by dragging a dispenser with manual<br>control. The membrane was then incubated with 2% BSA and<br>0.05% (v/v) Tween-20 in PBS for 1 hour in order to block the<br>membrane from making unwanted binding. The membrane was<br>rinsed with PBS containing 0.05% (v/v) Tween-20 to remove the<br>excess blocking reagent and left to dry at room temperature.<br>3. Results and discussion<br>A lateral flow immunosensor was developed by assembling<br>a lateral flow strip with sample application, detection, and<br>absorbent pads. The specific binding reaction was based on<br>immobilized Protein A and human IgG that was detected by the<br>conjugated MWCNT labels. Since CNTs have inherent affinity<br>to biological elements causing non-specific binding, control tests<br>were conducted at different functionalization steps. First, the<br>PVP treated MWCNTs were applied to the LF immunosensors<br>to bind with immobilized Protein A. As a result, unwanted<br>binding of the MWCNTs with Protein A at the capture zone was<br>observed as shown in Fig. 2(a). This non-specific binding could<br>have been caused by the affinity of the exposed MWCNTs<br>surface to the Protein A. Next, the crosslinking agents, EDC and<br>sulfo-NHS, were added to the PVP treated carbon nanotubes<br>and the same test was repeated. In this case, no binding reaction<br>was observed between the MWCNTs and Protein A at the<br>capture zone as shown in Fig. 2(b). This result showed that the<br>surfaces of the MWCNTs were completely covered by the PVP<br>and the amide coupling agents limiting unoccupied sites from<br>non-specific binding. Finally, MWCNT–IgG conjugate solution<br>was applied, where binding between human IgG and Protein A<br>was evident at the capture zone as shown in Fig. 2(c). Scanning<br>

electron microscopy (SEM) was used to examine the capture<br>zone, where the MWCNTs formed a conducting network as<br>a result of binding. Fig. 3 shows SEM image of the nitrocellulose<br>membrane without MWCNT labels and with varying concen-<br>tration of MWCNT labels corresponding to the amount of<br>binding.<br>Prior to the immunoassay test, varying concentration of the<br>conjugate solutions ranging from 0 to 200 μg mlÀ1 was prepared<br>by dilution in PBS buffer in order to determine the detection limit<br>of the conjugate solution. 50 μl of each solution were applied to<br>the LF immunosensors where the conjugated human IgG was<br>captured by the Protein A at the capture zone resulting in<br>colorimetric signal as shown in Fig. 4. As depicted in Fig. 4, the<br>color intensity of the signal was stronger for higher concentration<br>of the conjugated human IgG. For quantitative analysis of the<br>binding reaction, the resistance across the capture line was<br>measured after the complete development of the visible signal<br>($15 minutes). The measured electrical resistance decreased as<br>the concentration of the MWCNT–human IgG conjugate<br>increased due to the increased amount of MWCNTs forming<br>a conducting network. Three replications of the immunosensors<br>with same conjugate concentration were tested for consistency of<br>the measurements. The electrical resistance was measured using<br>electrodes with 3 mm spacing and while maintaining same<br>contact resistance.<br>The developed immunosensor was used with increasing<br>concentration of human IgG as target analyte in solution.<br>Concentration of the human IgG target analyte ranged from 25<br>to 200 μg mlÀ1 and the concentration of the conjugate solution<br>was fixed at 200 μg mlÀ1. Each target analyte solution was briefly<br>mixed with the conjugate and then the mixture was applied to the<br>LF immunosensor as a one-step immunoassay process. As the<br>sample solution moved across the capture zone, a competitive<br>immunoassay reaction between the conjugated human IgG and<br>those in the solution occurred. This produced a detection signal<br>by the MWCNTs across the capture line varying in color<br>intensity that was inversely proportional to the amount of<br>human IgG target analyte captured as shown in Fig. 5(a). The<br>detection signals were visible with the application of only 20 μl,<br>and saturation occurred with the application of 50 μl of total<br>sample solutions within 15 minutes. The visibility of the capture<br>zone decreased with increasing target human IgG concentration.<br>As the concentration of the target analyte increased, it occupied<br>more binding sites limiting the binding reaction between the<br>conjugated human IgG and the Protein A. The corresponding<br>measured results in Fig. 5(b) clearly show that the electrical<br>resistance increased with increasing concentration of the target<br>human IgG. Although the visibility of capture lines decreased with<br>increasing target IgG concentration (100, 150, and 200 μg mlÀ1)<br>

<br>Quantitative lateral flow immunosensor using carbon nanotubes as label<br>Adeyabeba Abera and Jin-Woo Choi*<br>Received 28th June 2010, Accepted 26th August 2010<br>DOI: 10.1039/c0ay00412j<br>A novel immunosensor utilizing multi-walled carbon nanotube as label on a lateral flow system for<br>simple and quantitative electrical detection was investigated. Multi-walled carbon nanotubes<br>(MWCNTs) were first modified with polyvinylpyrrolidone (PVP) for uniform dispersion in aqueous<br>solution. Then, the MWCNTs were conjugated with human immunoglobulin G (IgG) using 1-(3-<br>(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC) coupling chemistry. The lateral<br>flow immunosensor was made of nitrocellulose membrane that transports sample and reagents through<br>a porous structure by capillary action. The performance of the immunosensor was demonstrated using<br>human IgG as a model analyte competing with the conjugate (MWCNT-labeled human IgG) at the<br>capture zone. As a result of binding reaction between the conjugate and the immobilized Protein A at<br>the capture zone, the conjugated MWCNTs formed a conducting network at the capture zone<br>providing conductance measurement corresponding to the amount of captured conjugate. Quantitative<br>immunoassay response for the target human IgG was demonstrated in the range of 25 to 200 μg mlÀ1<br>without an additional amplification step. The presented immunosensing technique could be expanded<br>for the detection of various analyte-specific biomolecules with a potential for simple and rapid tests<br>suitable for point-of-care diagnostics.<br>1. Introduction<br>Lateral flow (LF) immunoassay is one of the most successful<br>point-of-care diagnostic tools commercially available for detecting<br>various health and environmental agents.1–6<br>A lateral flow<br>immunosensor is made of a porous membrane for both immobi-<br>lizing biological receptors and transporting sample reagents.<br>Sample reagents are transported in the lateral direction through<br>the porous membrane by capillary force and captured by the<br>biological receptors immobilized at the capture zone. LF immu-<br>nosensors are simple to use, low-cost, rapid, and portable with<br>relatively long shelf life. Examples of lateral flow diagnostics<br>include home pregnancy tests, detection of drugs of abuse, diag-<br>nosis of infectious diseases, and cancer markers. However, such<br>systems often suffer from low sensitivity, and are mostly restricted<br>to qualitative or semi-quantitative measurements.<br>Generally, LF immunosensors are based on optical detection<br>where tagged particles or enzymes provide visible signal generation<br>or amplification. Typically, colloidal gold is used as a label for<br>colorimetric detection traced by the visible color. Several efforts<br>have been made towards signal quantification using enhanced<br>colorimetric labels or conducting materials such as phosphorescent<br>nanoparticles,7 polyaniline,8 and polyaniline with colloidal gold.9<br>Colorimetric techniques are often limited by low sensitivity while<br>fluorescent techniques suffer from instability, photobleaching,<br>activation step, and requirement for a complicated reader device.<br>This work addresses such limitations by utilizing carbon<br>nanotubes (CNTs) as labeling material for direct electrical signal<br>measurement. CNTs have remarkable one-dimensional elec-<br>tronic conduction properties and large surface area, and allow<br>the adsorption of biomolecules for sensing.10–12 In order to utilize<br>these properties of carbon nanotubes for biosensing applications,<br>surface functionalization is necessary to uniformly disperse<br>CNTs in aqueous solution and immobilize biorecognition<br>molecules on the surface of the nanotubes. Adsorption of<br>surfactants and polymers on the surface of CNTs is considered as<br>an effective method to enhance the dispersion of CNTs without<br>affecting their properties.13–15 Compared to CNTs, other con-<br>ducting materials such as colloidal gold and polymer nanowires<br>do not possess the unique material properties suitable for surface<br>functionalization or maintain high electrical conduction after<br>binding reaction.9,16<br>In this work, a new immunoassay technique using carbon<br>nanotube labels on lateral flow system is introduced. The illus-<br>tration of this technique is shown in Fig. 1. MWCNTs are<br>dispersed in water with PVP using sonication. Adsorption of<br>PVP provides a thin and rough layer of organic coating by<br>wrapping around the MWCNTs. Human IgG is immobilized on<br>the bare (not covered by PVP) surface of the MWCNTs. The<br>MWCNT–human IgG conjugate is then applied on a lateral flow<br>immunosensor where the MWCNT–human IgG conjugate is to<br>be captured by immobilized Protein A at the capture zone. The<br>conjugated MWCNTs form a conducting network at the capture<br>zone corresponding to the amount of binding. Consequently,<br>quantitative signal can be obtained by simple resistance<br>measurement of the capture zone. Blocking schemes were also<br>implemented on the MWCNTs and the lateral flow strips to<br>address non-specific interaction.<br>2. Methods and experiments<br>2.1 Materials and reagents<br>Carboxylated multi-walled carbon nanotubes (purity &gt; 95 wt%)<br>were obtained from Cheaptubes, Inc. (Brattleboro, VT). Hi-flow<br>Department of Electrical and Computer Engineering, Louisiana State<br>University, Baton Rouge, LA, 70803, USA; Web: www.ece.lsu.<br>edu/biomems/. E-mail: choi@ece.lsu.edu; Tel: +1-225-578-8764<br>This journal is © The Royal Society of Chemistry 2010<br>Anal. Methods, 2010, 2, 1819–1822 | 1819<br>TECHNICAL NOTE<br>www.rsc.org/methods | Analytical Methods<br>Downloaded on 30 December 2012<br>Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J<br>View Article Online / Journal Homepage / Table of Contents for this issue<br>Page 2<br>Plus nitrocellulose membranes (HF13502), glass fiber pads,<br>cellulose membranes, and a supporting card for lateral flow assay<br>were obtained from Millipore (Bedford, MA). Protein A sus-<br>pended in solution was obtained from BioVision, Inc. (Mountain<br>View, CA). Purified human immunoglobulin G (IgG, 6.2 mg mlÀ1)<br>suspended in 0.01 M sodium phosphate (0.15 M NaCl, pH 7.4)<br>was obtained from Sigma-Aldrich (St Louis, MO). Poly-<br>vinylpyrrolidone (PVP, Mw 10 000), bovine serum albumin (BSA),<br>Tween-20, glutaraldehyde, and 2-(N-morpholino)ethanesulfonic<br>acid (MES) were from Sigma-Aldrich. 1-(3-(Dimethylamino)-<br>propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-<br>hydroxysulfosuccinimide (sulfo-NHS) were obtained from<br>Thermo Fisher Scientific, Inc. (Rockford, IL). All immunore-<br>agents were diluted in pH 7.4 phosphate saline buffer (PBS) unless<br>noted otherwise.<br>2.2 MWCNT–antibody conjugation<br>In order to start the conjugation process, 1.5 mg of carboxylated<br>MWCNTs were dispersed in 2 ml of deionized (DI) water with 0.6<br>mg of PVP. The solution was sonicated for 4 hours in an ultra-<br>sonic bath at 27 C to exfoliate the MWCNTs and accelerate the<br>dispersion. The dispersed solution was mixed with 1 ml of EDC<br>(0.4 M) and sulfo-NHS (0.1 M) in pH 6.1 MES buffer. The<br>solution was left to react for 30 minutes with gentle agitation at<br>room temperature. The mixture was microcentrifuged at 13 000<br>rpm for 3 minutes with PBS buffer and the supernatant was dis-<br>carded. The washing and re-suspending process was repeated<br>three times to remove excess reagents. Human IgG solution was<br>prepared at 200 μg mlÀ1 by diluting with PBS buffer for conju-<br>gation. The solution containing functionalized MWCNTs was<br>then mixed with 100 μl of the human IgG solution and left to react<br>overnight at 4 C. This mixture was centrifuged at 13 000 rpm for<br>3 minutes and the supernatant was discarded. The washing and re-<br>suspending process was repeated three times to remove unbound<br>human IgG, and the resulting MWCNT–antibody conjugate<br>solution was kept at 4 C until use.<br>2.3 Preparation of lateral flow immunosensors<br>A nitrocellulose membrane was used as a detection pad with<br>designated capture zone for immobilizing Protein A. Fiber glass<br>and cellulose membranes were used for sample application and<br>for absorption of excess reagents by placing them at the two ends<br>of the nitrocellulose membrane, respectively. The LF immuno-<br>strips were prepared following general procedure reported else-<br>where.7,9 First, a nitrocellulose membrane was saturated with<br>10% (v/v) methanol in deionized (DI) water for 30 minutes to<br>remove any residue and dried in air at room temperature. The<br>membrane was then treated with 0.5% (v/v) glutaraldehyde<br>solution to strengthen the immobilization of Protein A on the<br>nitrocellulose membrane and rinsed with DI water. After drying,<br>Protein A diluted in PBS buffer to 1 mg mlÀ1 was immobilized at<br>a designated capture zone by dragging a dispenser with manual<br>control. The membrane was then incubated with 2% BSA and<br>0.05% (v/v) Tween-20 in PBS for 1 hour in order to block the<br>membrane from making unwanted binding. The membrane was<br>rinsed with PBS containing 0.05% (v/v) Tween-20 to remove the<br>excess blocking reagent and left to dry at room temperature.<br>3. Results and discussion<br>A lateral flow immunosensor was developed by assembling<br>a lateral flow strip with sample application, detection, and<br>absorbent pads. The specific binding reaction was based on<br>immobilized Protein A and human IgG that was detected by the<br>conjugated MWCNT labels. Since CNTs have inherent affinity<br>to biological elements causing non-specific binding, control tests<br>were conducted at different functionalization steps. First, the<br>PVP treated MWCNTs were applied to the LF immunosensors<br>to bind with immobilized Protein A. As a result, unwanted<br>binding of the MWCNTs with Protein A at the capture zone was<br>observed as shown in Fig. 2(a). This non-specific binding could<br>have been caused by the affinity of the exposed MWCNTs<br>surface to the Protein A. Next, the crosslinking agents, EDC and<br>sulfo-NHS, were added to the PVP treated carbon nanotubes<br>and the same test was repeated. In this case, no binding reaction<br>was observed between the MWCNTs and Protein A at the<br>capture zone as shown in Fig. 2(b). This result showed that the<br>surfaces of the MWCNTs were completely covered by the PVP<br>and the amide coupling agents limiting unoccupied sites from<br>non-specific binding. Finally, MWCNT–IgG conjugate solution<br>was applied, where binding between human IgG and Protein A<br>was evident at the capture zone as shown in Fig. 2(c). Scanning<br>Fig. 1 Illustration of the detection principle using carbon nanotubes as label for quantitative lateral flow immunosensing in competitive format. Carbon<br>nanotubes form a conducting network at the capture zone corresponding to the amount of binding reaction. Consequently, quantitative signal can be<br>obtained by simple resistance measurement of the capture zone.<br>1820 | Anal. Methods, 2010, 2, 1819–1822<br>This journal is © The Royal Society of Chemistry 2010<br>Downloaded on 30 December 2012<br>Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J<br>View Article Online<br>Page 3<br>electron microscopy (SEM) was used to examine the capture<br>zone, where the MWCNTs formed a conducting network as<br>a result of binding. Fig. 3 shows SEM image of the nitrocellulose<br>membrane without MWCNT labels and with varying concen-<br>tration of MWCNT labels corresponding to the amount of<br>binding.<br>Prior to the immunoassay test, varying concentration of the<br>conjugate solutions ranging from 0 to 200 μg mlÀ1 was prepared<br>by dilution in PBS buffer in order to determine the detection limit<br>of the conjugate solution. 50 μl of each solution were applied to<br>the LF immunosensors where the conjugated human IgG was<br>captured by the Protein A at the capture zone resulting in<br>colorimetric signal as shown in Fig. 4. As depicted in Fig. 4, the<br>color intensity of the signal was stronger for higher concentration<br>of the conjugated human IgG. For quantitative analysis of the<br>binding reaction, the resistance across the capture line was<br>measured after the complete development of the visible signal<br>($15 minutes). The measured electrical resistance decreased as<br>the concentration of the MWCNT–human IgG conjugate<br>increased due to the increased amount of MWCNTs forming<br>a conducting network. Three replications of the immunosensors<br>with same conjugate concentration were tested for consistency of<br>the measurements. The electrical resistance was measured using<br>electrodes with 3 mm spacing and while maintaining same<br>contact resistance.<br>The developed immunosensor was used with increasing<br>concentration of human IgG as target analyte in solution.<br>Concentration of the human IgG target analyte ranged from 25<br>to 200 μg mlÀ1 and the concentration of the conjugate solution<br>was fixed at 200 μg mlÀ1. Each target analyte solution was briefly<br>mixed with the conjugate and then the mixture was applied to the<br>LF immunosensor as a one-step immunoassay process. As the<br>sample solution moved across the capture zone, a competitive<br>immunoassay reaction between the conjugated human IgG and<br>those in the solution occurred. This produced a detection signal<br>by the MWCNTs across the capture line varying in color<br>intensity that was inversely proportional to the amount of<br>human IgG target analyte captured as shown in Fig. 5(a). The<br>detection signals were visible with the application of only 20 μl,<br>and saturation occurred with the application of 50 μl of total<br>sample solutions within 15 minutes. The visibility of the capture<br>zone decreased with increasing target human IgG concentration.<br>As the concentration of the target analyte increased, it occupied<br>more binding sites limiting the binding reaction between the<br>conjugated human IgG and the Protein A. The corresponding<br>measured results in Fig. 5(b) clearly show that the electrical<br>resistance increased with increasing concentration of the target<br>human IgG. Although the visibility of capture lines decreased with<br>increasing target IgG concentration (100, 150, and 200 μg mlÀ1),<br>Fig. 2 Non-specific adsorption on the nitrocellulose membrane: (a)<br>MWCNTs partially coated with PVP were captured by Protein A due to<br>non-specific adsorption which must be addressed to avoid false signal; (b)<br>almost no detection signal was observed at the capture zone indicating<br>that the surface of the MWCNTs were completely covered eliminating<br>non-specific adsorption, and (c) selective binding between the MWCNT–<br>human IgG conjugate and immobilized Protein A.<br>Fig. 3 SEM images of the MWCNT network at the capture zone: (a) before sample application; (b) lower MWCNT coverage due to a smaller number<br>of binding; and (c) higher MWCNT coverage due to a larger number of binding. The scale bar is 1 μm.<br>Fig. 4 Calibration results using conjugate solutions in different<br>concentrations. MWCNTs as label provided a quantitative detection<br>capability.<br>This journal is © The Royal Society of Chemistry 2010<br>Anal. Methods, 2010, 2, 1819–1822 | 1821<br>Downloaded on 30 December 2012<br>Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J<br>View Article Online<br>Page 4<br>the electrical measurements clearly showed differences corre-<br>sponding to the various concentrations at all levels.<br>4. Conclusions<br>A lateral flow immunosensing technique using carbon nanotubes<br>as a label was effectively demonstrated for quantitative antibody<br>detection based on simple electrical measurements. The surface<br>of the MWCNTs was partially modified with PVP for uniform<br>dispersion in aqueous solutions. Further functionalization steps<br>were conducted to attach antibodies on the exposed surface of<br>MWCNTs utilizing EDC/sulfo-NHS chemistry. The modified<br>MWCNTs successfully provided both direct colorimetric and<br>conductimetric measurements for antibody binding without addi-<br>tion amplification step. SEM images showed the MWCNT<br>network formed as a result of antibody binding at the capture zone.<br>The lateral flow system facilitated the binding reaction and reagent<br>handling in a simple and convenient manner. This immunosensing<br>technique is easy for integration and requires ordinary tools for<br>signal measurement. Therefore, it holds the potential for quanti-<br>tative determination of various antibodies and antigen in a point-<br>of-care setup. The detection limit and time could be improved<br>further with proper optimization of the conjugating process and<br>the lateral flow system<br>

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[[Category:Lateral_Flow]]