Biosensors and Bioelectronics 72 (2015) 156–159
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
An ultrasensitive squamous cell carcinoma antigen biosensing platform utilizing double-antibody single-channel amplification strategy Xiang Ren a, Dan Wu a, Yuhuan Wang a, Yunhui Zhang a, Dawei Fan a, Xuehui Pang a, Yueyun Li b, Bin Du a, Qin Wei a,n a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b School of Chemical Engineering, Shandong University of Technology, Zibo 255049, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 31 January 2015 Received in revised form 14 April 2015 Accepted 6 May 2015 Available online 7 May 2015
A novel electrochemical immunosensor was developed for ultrasensitive detection of squamous cell carcinoma antigen (SCCA), which was based on the double-antibody single-channel amplification strategy. For the first time, human immunoglobulin antibody (anti-HIgG) was used as the supporting framework to amplify the loading quantity of SCCA antibody (anti-SCCA). In this strategy, SCCA can be detected without using mesoporous nanometers to amplify the signal. In addition, Pd icosahedrons were first used as the connecter to immobilize the antibodies and strengthen the sensitivity. Only one touch point exists under the limited condition between a sphere and another shape in geometry, thus the Pd icosahedron is an excellent candidate as the role of connecter. Gold nanoparticles (Au NPs) decorated with mercapto-functionalized graphene sheets (Au@GS) were synthesized as the transducing materials. The fabricated immunosensor exhibited an excellent detection limit of 2.8 pg/mL and wide linear range of 0.01–5 ng/mL. This kind of immunosensor would provide a potential application in clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: Double-antibody single-channel strategy Pd icosahedron Squamous cell carcinoma antigen Mercapto-functionalized graphene sheets Immunosensor
1. Introduction Squamous cell carcinoma antigen (SCCA) is a kind of tumor antigen TA-4 separated from a cervical squamous cell carcinoma (SCC) (Takeuchi et al., 2003). The morbidity of cervical SCC increases from 12% to more than 90% with elevated circulating levels of SCCA (Kato, 1992). It is regarded as a sensitive tumor marker in patients with cervical cancer. Until now, several techniques have been developed in its detection, such as common sandwich immunosensor (Wu et al., 2013), enzyme-linked immunosensor assay (Erickson et al., 2010), and chemiluminescence immunoassay (Li et al., 2014b). All of these methods employ mesoporous materials to amplify signals with complex processes. Thus, a new strategy for the marker detection is necessary in the clinical test and therapeutic evaluation. Immunoassay is an effective method in tumor marker detection (Ren et al., 2014a, 2014b). But the amplification strategy is mainly based on using the mesoporous nanomaterials (Lei and Ju, 2012; Wu et al., 2012; Zhang et al., 2011a) to enlarge the capacity of biomolecules and enhance signals (Bi et al., 2013; Jeong et al., 2013; Qu et al., 2014). n
Corresponding author. Fax: þ86 531 8276 5969. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2015.05.012 0956-5663/& 2015 Elsevier B.V. All rights reserved.
In this research, the double-antibody single-channel immunosensor was fabricated to realize the marker detection, which employed human immunoglobulin antibody (anti-HIgG) instead of mesoporous nanomaterials acting as the supporting framework to amplify the loading quantity of SCCA antibody (anti-SCCA) for the first time. In this strategy, the immunosensor would be able to achieve excellent properties by the following reasons. First, the immuno-active materials own better recognition capability to ensure the sensor specific recognition (Kiefel et al., 1987). Second, biomolecules have prominent hydrophilcity to form homogeneous solution than nanomaterials (Cheng and Rossky, 1998). Third, the size of antibody or antigen is about 10–15 nm which is much smaller than that of 100 nm (the traditional size of nanomaterials applied in immunosensor), so the nanomaterials (large scale) may not obtain a satisfied result due to an inadequate adsorption. Several smaller sized nanomaterials have been applied in the immunosensor in recent years (Feng et al., 2012a). Thus, the double-antibody single-channel immunosensor is an excellent candidate method to detect tumor markers. In this research, gold nanoparticles (Au NPs) decorated with mercapto-functionalized graphene sheets (Au@GS) were
X. Ren et al. / Biosensors and Bioelectronics 72 (2015) 156–159
synthesized to increase the fixing quantity of anti-HIgG. Pd icosahedrons were first used in immunosensor to amplify the antigen detection quantity. In geometry, typically, only one touch point exists between a sphere and another shape under the limited condition. In the area of immunosensor, spherical materials (such as noble metal, mesoporous materials, quantum dots) have been applied in the sensor frequently (Feng et al., 2012b; Li et al., 2014a; Zhang et al., 2011b). However, the point-of-touching may exhibit lower binding force than the spot-of-touching. The Pd icosahedrons own twenty spots and every spot may contact the biomolecules more tightly than the point-of-touching of nanosphere.
2. Experimental In this work, mercapto-functionalized graphene sheets were synthesized to capture more Au NPs. And the testing system was three-electrode configuration (ESI†). The glassy carbon electrode (GCE) was polished with a series of alumina powders (1, 0.3, 0.05 μm) and washed with water. Afterwards, the Au@GS modified electrode could adsorb more anti-HIgGs to protect and strengthen the framework and enlarge the capacity of HIgG. Once BSA blocked the non-specific binding sites (other binding sites other than HIgG), HIgG would be incubated on the anti-HIgG subsequently. Then, a certain proportion of antibodies (anti-HIgG: antiSCCA ¼2:8) were incubated with Pd icosahedrons. The antibody incubation compound anti-HIgG@Pd@anti-SCCA (AbS@Pd@AbH) was blocked with BSA as well. And AbS@Pd@AbH was incubated on the former layer. After the SCCA was captured on the electrode, the sensor was fabricated well (Fig. 1) (detailed in ESI†). The signal could be achieved by the solution of K3Fe(CN)6 (4 mM) and KNO3 (0.1 M).
3. Results and discussion GS–SH is synthesized according to a reported method (Wang et al., 2014) with some modification for the first time in this strategy (ESI†). The Au NPs are connected on the GS–SH sufficiently. From Fig. 2A, it can be clearly seen that the GS is silk-like and the morphology is straticulate. Au@GS is shown in Fig. 2B. In the margin and middle of the GS, there exist some brilliant points
Fig. 1. Fabrication of the double-antibody single-channel immunosensor for SCCA detection.
157
(a, c) and relative tint points (b, d). These points are all Au NPs indicating the GS is straticulate and the Au NPs are connected on GS uniformly. Pd icosahedrons are used as the connecter between anti-HIgG and anti-SCCA. And the preparation of Pd icosahedrons is shown in ESI†. Fig. 3A is the SEM image of Pd icosahedrons at low magnification. It displays that the Pd nanoparticles are dispersed well and the size was uniform. Fig. 3B is the TEM image of Pd icosahedrons, and it can be seen obviously that the icosahedron was synthesized well compared with the inset of Fig. 3B which is an icosahedron in 3D image. The size of Pd icosahedrons is about 40 nm in diameter and the antibody is about 10–15 nm. This is a balanced and appropriate size, indicating the combination of AbS@Pd@AbH was effective. A Rubik's cube theory is proposed and that may be explained as follows. First, the connecter (Pd icosahedron) is in the same order of magnitude with the antibodies in size, and the incubation (AbS@Pd@AbH) can be prepared well like a 64 Rubik's cube (ESI†, Fig. S2). The inside middle box (8 cubic) is like the Pd icosahedron, and the outside 56 cubes are like the antibodies, which can realize many times amplification. Second, if the connecter is smaller than the size of antibody (the connecter and antibody are not in a comparative size), the antibodies cannot be combined well on the particles due to the limitation of size. Third, if the connecter is much larger, for example, over a hundred nanometers, the connecter is many times larger than antibody in size which may leave over many nonspecific active sites resulting in the inaccuracy of the results. In addition, much larger particles may not exhibit a better testing result due to the bad film-forming property. Thus, the Pd icosahedron is the better candidate as a connecter in the double-antibody single-channel amplification strategy. In this research, a SCCA sensor was developed. K3[Fe(CN)6] was used as the signal source. In order to realize better detecting effect, optimization of the experimental conditions was conducted. The ratio of antibodies, pH values, concentration of base solution (K3[Fe(CN)6]) and concentration of base material (Au@GS) were investigated in this study (ESI†, Fig. S3). The obtained results are anti-HIgG: anti-SCCA ¼ 2:8, pH ¼7.4, CK3[Fe(CN)6] ¼4 mg/mL, CAu@GS ¼1.5 mg/mL respectively. Under the optimum conditions, immunosensor was used to detect different concentrations of SCCA. The square wave voltammetry data were shown in Fig. S4 (ESI†). It was shown in Fig. 4 the analysis and detection of SCCA with prepared sensor were satisfactory. It worked well over a broad liner range of 0.01–5 ng/mL with a low detection limit of 2.8 pg/mL at a signal-to-noise ratio of 3s (where s is the standard deviation of a blank solution, n ¼11). The equation was ΔI ¼3.22 log C þ7.96, r¼ 0.9968. The selectivity of the sensor plays an essential role in the analysis of biological samples. Several other tumor markers were detected in the selectivity research. As shown in Fig. S5 (ESI†), the SCCA-free immunosensors (20 ng/mL CEA, AFP and CA125 respectively) exhibited the similar current change which approached to zero. The other four sensors exhibited similar current change which indicated the sensor was not disturbed by other tumor markers. The selectivity of the immunosensor was acceptable. Stability is another considered factor in potential practical application. Several fabricated immunosensors were stored in refrigerator at 4 °C. A week later, the signal strength decreased to 95% of the initial value, and half a month later, it decreased to 88% of initial value (Fig. S7), indicating the result was satisfactory. Reproducibility was also studied in this immunosensor fabrication. 5 prepared sensors were tested under identical conditions (Fig. S6). The RSD of the measurement was within 2.7%, suggesting the precision of the biosensor was reasonably good for SCCA detection. To evaluate the performance of the novel immunosensor,
158
X. Ren et al. / Biosensors and Bioelectronics 72 (2015) 156–159
Fig. 2. SEM images of GS–SH (A) and Au@GS (B).
Fig. 3. (A) SEM image of Pd icosahedrons at low magnification; and (B) TEM image of Pd icosahedrons.
Fig. 4. Calibration curve of the prepared double-antibody single-channel sensor, error bar¼ RSD (n ¼5).
X. Ren et al. / Biosensors and Bioelectronics 72 (2015) 156–159
Table 1 Results for the detection of SCCA in human serum sample. Human serum sample (ng/mL)
Addition content (ng/mL)
Detection content (ng/mL)
RSD (%, n ¼5)
0.53
1 2
1.51 70.04 2.50 70.06
2.83 4.65
98.5 97.1
1 2
1.28 70.06 2.3370.05
1.96 3.14
98.1 101.6
0.29
Recovery (%)
human serum samples were tested in this research to verify the precision. The RSD was ranged from 1.96% to 4.65%, and the recovery of the sensor was from 97.1% to 101.6% (Table 1). In order to verify the amplification property of the double-antibody single-channel amplification strategy, some other tumor marker detection methods have been compared with this proposed strategy (Table S2). And in this research, SCCA was detected with a satisfactory result in comparison with some reported researches (Table S1).
4. Conclusion In this research, a double-antibody single-channel amplification strategy was adopted to develop a novel immunosensor for the detection of SCCA. Pd icosahedrons were used as the connecter of anti-SCCA and anti-HIgG, which might increase the captured quantity of SCCA to realize a lower detection. Au@GS was applied in the fabrication to enlarge the captured biomolecules. The prepared immunosensor exhibited excellent properties which would provide a potential application in clinical diagnose.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, 21377046, 21405059), the Science and Technology Plan Project of Jinan (No.
159
201307010), The Science and Technology Development Plan of Shandong Province (No. 2014GSF120004). And Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and University of Jinan (No. ts20130937).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.05.012.
References Bi, S., Zhao, T., Luo, B., Zhu, J.-J., 2013. Chem. Commun. 49, 6906–6908. Cheng, Y.-K., Rossky, P.J., 1998. Nature 392, 696–699. Erickson, J.A., Lu, J., Smith, J.J., Bornhorst, J.A., Grenache, D.G., Ashwood, E.R., 2010. Clin. Chem. 56, 1496–1499. Feng, L.-N., Bian, Z.-P., Peng, J., Jiang, F., Yang, G.-H., Zhu, Y.-D., Yang, D., Jiang, L.-P., Zhu, J.-J., 2012a. Anal. Chem. 84, 7810–7815. Feng, L.-N., Peng, J., Zhu, Y.-D., Jiang, L.-P., Zhu, J.-J., 2012b. Chem. Commun. 48, 4474–4476. Jeong, B., Akter, R., Han, O.H., Rhee, C.K., Rahman, M.A., 2013. Anal. Chem. 85, 1784–1791. Kato, H., 1992. Squamous cell carcinoma antigen. Serological Cancer Markers. Springer, pp. 437–451. Kiefel, V., Santoso, S., Weisheit, M., Mueller-Eckhardt, C., 1987. Blood 70, 1722–1726. Lei, J., Ju, H., 2012. Chem. Soc. Rev. 41, 2122–2134. Li, D., Li, J., Jia, X., Wang, E., 2014a. Electrochem. Commun. 42, 30–33. Li, X., Zhang, X., Ma, H., Wu, D., Zhang, Y., Du, B., Wei, Q., 2014b. Biosens. Bioelectron. 55, 330–336. Qu, F., Zhang, Y., Rasooly, A., Yang, M., 2014. Anal. Chem. 86, 973–976. Ren, X., Yan, T., Zhang, S., Zhang, X., Gao, P., Wu, D., Du, B., Wei, Q., 2014a. Analyst 139, 3061–3068. Ren, X., Yan, T., Zhang, Y., Wu, D., Ma, H., Li, H., Du, B., Wei, Q., 2014b. Biosens. Bioelectron. 58, 345–350. Takeuchi, S., Nonaka, M., Kadokura, M., Takaba, T., 2003. Ann. Thorac. Cardiovasc. Surg. 9, 98–104. Wang, Y., Zhang, Y., Su, Y., Li, F., Ma, H., Li, H., Du, B., Wei, Q., 2014. Talanta 124, 60–66. Wu, D., Fan, H., Li, Y., Zhang, Y., Liang, H., Wei, Q., 2013. Biosens. Bioelectron. 46, 91–96. Wu, D., Li, R., Wang, H., Liu, S., Wang, H., Wei, Q., Du, B., 2012. Analyst 137, 608–613. Zhang, J., Lei, J., Pan, R., Leng, C., Hu, Z., Ju, H., 2011a. Chem. Commun. 47, 668–670. Zhang, X., Li, S., Jin, X., Zhang, S., 2011b. Chem. Commun. 47, 4929–4931.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
An ultrasensitive squamous cell carcinoma antigen biosensing platform utilizing double-antibody single-channel amplification strategy Xiang Ren a, Dan Wu a, Yuhuan Wang a, Yunhui Zhang a, Dawei Fan a, Xuehui Pang a, Yueyun Li b, Bin Du a, Qin Wei a,n a Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China b School of Chemical Engineering, Shandong University of Technology, Zibo 255049, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 31 January 2015 Received in revised form 14 April 2015 Accepted 6 May 2015 Available online 7 May 2015
A novel electrochemical immunosensor was developed for ultrasensitive detection of squamous cell carcinoma antigen (SCCA), which was based on the double-antibody single-channel amplification strategy. For the first time, human immunoglobulin antibody (anti-HIgG) was used as the supporting framework to amplify the loading quantity of SCCA antibody (anti-SCCA). In this strategy, SCCA can be detected without using mesoporous nanometers to amplify the signal. In addition, Pd icosahedrons were first used as the connecter to immobilize the antibodies and strengthen the sensitivity. Only one touch point exists under the limited condition between a sphere and another shape in geometry, thus the Pd icosahedron is an excellent candidate as the role of connecter. Gold nanoparticles (Au NPs) decorated with mercapto-functionalized graphene sheets (Au@GS) were synthesized as the transducing materials. The fabricated immunosensor exhibited an excellent detection limit of 2.8 pg/mL and wide linear range of 0.01–5 ng/mL. This kind of immunosensor would provide a potential application in clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: Double-antibody single-channel strategy Pd icosahedron Squamous cell carcinoma antigen Mercapto-functionalized graphene sheets Immunosensor
1. Introduction Squamous cell carcinoma antigen (SCCA) is a kind of tumor antigen TA-4 separated from a cervical squamous cell carcinoma (SCC) (Takeuchi et al., 2003). The morbidity of cervical SCC increases from 12% to more than 90% with elevated circulating levels of SCCA (Kato, 1992). It is regarded as a sensitive tumor marker in patients with cervical cancer. Until now, several techniques have been developed in its detection, such as common sandwich immunosensor (Wu et al., 2013), enzyme-linked immunosensor assay (Erickson et al., 2010), and chemiluminescence immunoassay (Li et al., 2014b). All of these methods employ mesoporous materials to amplify signals with complex processes. Thus, a new strategy for the marker detection is necessary in the clinical test and therapeutic evaluation. Immunoassay is an effective method in tumor marker detection (Ren et al., 2014a, 2014b). But the amplification strategy is mainly based on using the mesoporous nanomaterials (Lei and Ju, 2012; Wu et al., 2012; Zhang et al., 2011a) to enlarge the capacity of biomolecules and enhance signals (Bi et al., 2013; Jeong et al., 2013; Qu et al., 2014). n
Corresponding author. Fax: þ86 531 8276 5969. E-mail address: [email protected] (Q. Wei).
http://dx.doi.org/10.1016/j.bios.2015.05.012 0956-5663/& 2015 Elsevier B.V. All rights reserved.
In this research, the double-antibody single-channel immunosensor was fabricated to realize the marker detection, which employed human immunoglobulin antibody (anti-HIgG) instead of mesoporous nanomaterials acting as the supporting framework to amplify the loading quantity of SCCA antibody (anti-SCCA) for the first time. In this strategy, the immunosensor would be able to achieve excellent properties by the following reasons. First, the immuno-active materials own better recognition capability to ensure the sensor specific recognition (Kiefel et al., 1987). Second, biomolecules have prominent hydrophilcity to form homogeneous solution than nanomaterials (Cheng and Rossky, 1998). Third, the size of antibody or antigen is about 10–15 nm which is much smaller than that of 100 nm (the traditional size of nanomaterials applied in immunosensor), so the nanomaterials (large scale) may not obtain a satisfied result due to an inadequate adsorption. Several smaller sized nanomaterials have been applied in the immunosensor in recent years (Feng et al., 2012a). Thus, the double-antibody single-channel immunosensor is an excellent candidate method to detect tumor markers. In this research, gold nanoparticles (Au NPs) decorated with mercapto-functionalized graphene sheets (Au@GS) were
X. Ren et al. / Biosensors and Bioelectronics 72 (2015) 156–159
synthesized to increase the fixing quantity of anti-HIgG. Pd icosahedrons were first used in immunosensor to amplify the antigen detection quantity. In geometry, typically, only one touch point exists between a sphere and another shape under the limited condition. In the area of immunosensor, spherical materials (such as noble metal, mesoporous materials, quantum dots) have been applied in the sensor frequently (Feng et al., 2012b; Li et al., 2014a; Zhang et al., 2011b). However, the point-of-touching may exhibit lower binding force than the spot-of-touching. The Pd icosahedrons own twenty spots and every spot may contact the biomolecules more tightly than the point-of-touching of nanosphere.
2. Experimental In this work, mercapto-functionalized graphene sheets were synthesized to capture more Au NPs. And the testing system was three-electrode configuration (ESI†). The glassy carbon electrode (GCE) was polished with a series of alumina powders (1, 0.3, 0.05 μm) and washed with water. Afterwards, the Au@GS modified electrode could adsorb more anti-HIgGs to protect and strengthen the framework and enlarge the capacity of HIgG. Once BSA blocked the non-specific binding sites (other binding sites other than HIgG), HIgG would be incubated on the anti-HIgG subsequently. Then, a certain proportion of antibodies (anti-HIgG: antiSCCA ¼2:8) were incubated with Pd icosahedrons. The antibody incubation compound anti-HIgG@Pd@anti-SCCA (AbS@Pd@AbH) was blocked with BSA as well. And AbS@Pd@AbH was incubated on the former layer. After the SCCA was captured on the electrode, the sensor was fabricated well (Fig. 1) (detailed in ESI†). The signal could be achieved by the solution of K3Fe(CN)6 (4 mM) and KNO3 (0.1 M).
3. Results and discussion GS–SH is synthesized according to a reported method (Wang et al., 2014) with some modification for the first time in this strategy (ESI†). The Au NPs are connected on the GS–SH sufficiently. From Fig. 2A, it can be clearly seen that the GS is silk-like and the morphology is straticulate. Au@GS is shown in Fig. 2B. In the margin and middle of the GS, there exist some brilliant points
Fig. 1. Fabrication of the double-antibody single-channel immunosensor for SCCA detection.
157
(a, c) and relative tint points (b, d). These points are all Au NPs indicating the GS is straticulate and the Au NPs are connected on GS uniformly. Pd icosahedrons are used as the connecter between anti-HIgG and anti-SCCA. And the preparation of Pd icosahedrons is shown in ESI†. Fig. 3A is the SEM image of Pd icosahedrons at low magnification. It displays that the Pd nanoparticles are dispersed well and the size was uniform. Fig. 3B is the TEM image of Pd icosahedrons, and it can be seen obviously that the icosahedron was synthesized well compared with the inset of Fig. 3B which is an icosahedron in 3D image. The size of Pd icosahedrons is about 40 nm in diameter and the antibody is about 10–15 nm. This is a balanced and appropriate size, indicating the combination of AbS@Pd@AbH was effective. A Rubik's cube theory is proposed and that may be explained as follows. First, the connecter (Pd icosahedron) is in the same order of magnitude with the antibodies in size, and the incubation (AbS@Pd@AbH) can be prepared well like a 64 Rubik's cube (ESI†, Fig. S2). The inside middle box (8 cubic) is like the Pd icosahedron, and the outside 56 cubes are like the antibodies, which can realize many times amplification. Second, if the connecter is smaller than the size of antibody (the connecter and antibody are not in a comparative size), the antibodies cannot be combined well on the particles due to the limitation of size. Third, if the connecter is much larger, for example, over a hundred nanometers, the connecter is many times larger than antibody in size which may leave over many nonspecific active sites resulting in the inaccuracy of the results. In addition, much larger particles may not exhibit a better testing result due to the bad film-forming property. Thus, the Pd icosahedron is the better candidate as a connecter in the double-antibody single-channel amplification strategy. In this research, a SCCA sensor was developed. K3[Fe(CN)6] was used as the signal source. In order to realize better detecting effect, optimization of the experimental conditions was conducted. The ratio of antibodies, pH values, concentration of base solution (K3[Fe(CN)6]) and concentration of base material (Au@GS) were investigated in this study (ESI†, Fig. S3). The obtained results are anti-HIgG: anti-SCCA ¼ 2:8, pH ¼7.4, CK3[Fe(CN)6] ¼4 mg/mL, CAu@GS ¼1.5 mg/mL respectively. Under the optimum conditions, immunosensor was used to detect different concentrations of SCCA. The square wave voltammetry data were shown in Fig. S4 (ESI†). It was shown in Fig. 4 the analysis and detection of SCCA with prepared sensor were satisfactory. It worked well over a broad liner range of 0.01–5 ng/mL with a low detection limit of 2.8 pg/mL at a signal-to-noise ratio of 3s (where s is the standard deviation of a blank solution, n ¼11). The equation was ΔI ¼3.22 log C þ7.96, r¼ 0.9968. The selectivity of the sensor plays an essential role in the analysis of biological samples. Several other tumor markers were detected in the selectivity research. As shown in Fig. S5 (ESI†), the SCCA-free immunosensors (20 ng/mL CEA, AFP and CA125 respectively) exhibited the similar current change which approached to zero. The other four sensors exhibited similar current change which indicated the sensor was not disturbed by other tumor markers. The selectivity of the immunosensor was acceptable. Stability is another considered factor in potential practical application. Several fabricated immunosensors were stored in refrigerator at 4 °C. A week later, the signal strength decreased to 95% of the initial value, and half a month later, it decreased to 88% of initial value (Fig. S7), indicating the result was satisfactory. Reproducibility was also studied in this immunosensor fabrication. 5 prepared sensors were tested under identical conditions (Fig. S6). The RSD of the measurement was within 2.7%, suggesting the precision of the biosensor was reasonably good for SCCA detection. To evaluate the performance of the novel immunosensor,
158
X. Ren et al. / Biosensors and Bioelectronics 72 (2015) 156–159
Fig. 2. SEM images of GS–SH (A) and Au@GS (B).
Fig. 3. (A) SEM image of Pd icosahedrons at low magnification; and (B) TEM image of Pd icosahedrons.
Fig. 4. Calibration curve of the prepared double-antibody single-channel sensor, error bar¼ RSD (n ¼5).
X. Ren et al. / Biosensors and Bioelectronics 72 (2015) 156–159
Table 1 Results for the detection of SCCA in human serum sample. Human serum sample (ng/mL)
Addition content (ng/mL)
Detection content (ng/mL)
RSD (%, n ¼5)
0.53
1 2
1.51 70.04 2.50 70.06
2.83 4.65
98.5 97.1
1 2
1.28 70.06 2.3370.05
1.96 3.14
98.1 101.6
0.29
Recovery (%)
human serum samples were tested in this research to verify the precision. The RSD was ranged from 1.96% to 4.65%, and the recovery of the sensor was from 97.1% to 101.6% (Table 1). In order to verify the amplification property of the double-antibody single-channel amplification strategy, some other tumor marker detection methods have been compared with this proposed strategy (Table S2). And in this research, SCCA was detected with a satisfactory result in comparison with some reported researches (Table S1).
4. Conclusion In this research, a double-antibody single-channel amplification strategy was adopted to develop a novel immunosensor for the detection of SCCA. Pd icosahedrons were used as the connecter of anti-SCCA and anti-HIgG, which might increase the captured quantity of SCCA to realize a lower detection. Au@GS was applied in the fabrication to enlarge the captured biomolecules. The prepared immunosensor exhibited excellent properties which would provide a potential application in clinical diagnose.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Nos. 21175057, 21375047, 21377046, 21405059), the Science and Technology Plan Project of Jinan (No.
159
201307010), The Science and Technology Development Plan of Shandong Province (No. 2014GSF120004). And Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and University of Jinan (No. ts20130937).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.05.012.
References Bi, S., Zhao, T., Luo, B., Zhu, J.-J., 2013. Chem. Commun. 49, 6906–6908. Cheng, Y.-K., Rossky, P.J., 1998. Nature 392, 696–699. Erickson, J.A., Lu, J., Smith, J.J., Bornhorst, J.A., Grenache, D.G., Ashwood, E.R., 2010. Clin. Chem. 56, 1496–1499. Feng, L.-N., Bian, Z.-P., Peng, J., Jiang, F., Yang, G.-H., Zhu, Y.-D., Yang, D., Jiang, L.-P., Zhu, J.-J., 2012a. Anal. Chem. 84, 7810–7815. Feng, L.-N., Peng, J., Zhu, Y.-D., Jiang, L.-P., Zhu, J.-J., 2012b. Chem. Commun. 48, 4474–4476. Jeong, B., Akter, R., Han, O.H., Rhee, C.K., Rahman, M.A., 2013. Anal. Chem. 85, 1784–1791. Kato, H., 1992. Squamous cell carcinoma antigen. Serological Cancer Markers. Springer, pp. 437–451. Kiefel, V., Santoso, S., Weisheit, M., Mueller-Eckhardt, C., 1987. Blood 70, 1722–1726. Lei, J., Ju, H., 2012. Chem. Soc. Rev. 41, 2122–2134. Li, D., Li, J., Jia, X., Wang, E., 2014a. Electrochem. Commun. 42, 30–33. Li, X., Zhang, X., Ma, H., Wu, D., Zhang, Y., Du, B., Wei, Q., 2014b. Biosens. Bioelectron. 55, 330–336. Qu, F., Zhang, Y., Rasooly, A., Yang, M., 2014. Anal. Chem. 86, 973–976. Ren, X., Yan, T., Zhang, S., Zhang, X., Gao, P., Wu, D., Du, B., Wei, Q., 2014a. Analyst 139, 3061–3068. Ren, X., Yan, T., Zhang, Y., Wu, D., Ma, H., Li, H., Du, B., Wei, Q., 2014b. Biosens. Bioelectron. 58, 345–350. Takeuchi, S., Nonaka, M., Kadokura, M., Takaba, T., 2003. Ann. Thorac. Cardiovasc. Surg. 9, 98–104. Wang, Y., Zhang, Y., Su, Y., Li, F., Ma, H., Li, H., Du, B., Wei, Q., 2014. Talanta 124, 60–66. Wu, D., Fan, H., Li, Y., Zhang, Y., Liang, H., Wei, Q., 2013. Biosens. Bioelectron. 46, 91–96. Wu, D., Li, R., Wang, H., Liu, S., Wang, H., Wei, Q., Du, B., 2012. Analyst 137, 608–613. Zhang, J., Lei, J., Pan, R., Leng, C., Hu, Z., Ju, H., 2011a. Chem. Commun. 47, 668–670. Zhang, X., Li, S., Jin, X., Zhang, S., 2011b. Chem. Commun. 47, 4929–4931.
