Ultrasensitive multiplexed protein biomarker detection based on electrochemical tag incorporated polystyrene spheres as label

Sensors and Actuators B 186 (2013) 768–773

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Ultrasensitive multiplexed protein biomarker detection based on electrochemical tag incorporated polystyrene spheres as label Ting Li a,1 , Bo Shu a,1 , Bin Jiang a , Lu Ding c , Haizhi Qi a,∗ , Minghui Yang b,∗ , Fengli Qu c a

Department of General Surgery, The Second Xiang-ya Hospital, Central South University, Changsha 410011, China College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China Shandong Provincial Key Laboratory of Life-organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165, China b c

a r t i c l e

i n f o

Article history: Received 8 May 2013 Received in revised form 24 June 2013 Accepted 29 June 2013 Available online 8 July 2013 Keywords: Multiplexed Electrochemical immunosensor Interleukin-6 Interleukin-17 Polystyrene nanosphere

a b s t r a c t Multiplexed electrochemical immunosensor was developed for ultrasensitive detection of interleukin-6 (IL-6) and interleukin-17 (IL-17) using electrochemical tag incorporated polystyrene sphere (PS) as label. The electrochemical tag contained in the PS, cadmium ion (Cd2+ ) or ferrocene (Fc), could be effectively detected by square wave voltammetry (SWV) after dissolving the PS with organic solvent to release the electrochemical tags onto electrode surface. With the immobilization of two different antibodies onto the PS surface, respectively, the position and intensity of the current peaks due to the two electrochemical tags could reflect the identity and level of the corresponding protein biomarkers. The significant amount of electrochemical tags incorporated into PS greatly enhanced the detection sensitivity. The proposed immunoassay exhibited high sensitivity and good selectivity for the detection of IL-6 and IL-17. The response current was linear to the logarithm concentrations of IL-6 and IL-17 in the range from 1 pg/mL to1 ng/mL and 2 pg/mL to 1 ng/mL, respectively. The detection limits of IL-6 and IL-17 were 0.5 and 1 pg/mL (S/N = 3). These results suggested that this electrochemical immunosensor would be widely applied for clinical screening of protein biomarkers. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The advancement in clinical diagnostics and disease treatment has aroused the need for the development of sensitive, rapid, and low cost methods for the detection of protein biomarkers [1–3]. Immunosensors based on specific antibody–antigen interaction is one of the most widely utilized analytical techniques in the quantitative detection of biomarkers [4–7]. Clinically, usually several protein biomarkers together will provide the most reliable information for the precise diagnosis of certain kind of diseases [8–10]. So compared to the traditional immunosensor that can only detect one protein at a time, a high throughput platform for the multiplexed quantification of proteins is of much greater interest, which can shorten analytical time and increase detection efficiency. To develop such multiplexed immunosensor platform, capture antibodies are usually immobilized onto separated electrode arrays and using a single label for signal transduction [11–13]. However, to prevent cross-talk between neighboring electrodes, there need to be enough spaces left between electrodes. Another alternative

∗ Corresponding authors. Tel.: +86 731 88836356; fax: +86 731 88836356. E-mail addresses: Qi [email protected] (H. Qi), [email protected] (M. Yang). 1 These authors contributed equally to this work. 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.06.104

method is to use multiple labels, each to a specific protein for signal transduction [14]. Among the different kinds of immunosensor transduction methods, such as fluorescence [15,16], surface plasmon resonance (SPR) [17,18], quartz crystal microbalance (QCM) [19,20] and electrochemistry, electrochemical immunosensors due to their high sensitivity, fast response, low cost and simple instrumentation have attracted significant research interest [21,22]. In addition, the electrochemical sensors can be easily miniaturized, which plus the above mentioned advantages enables them be easily developed into point-of-care diagnostic devices. To prepare the electrochemical immunosensor and achieve sensitive detection, various electrochemical tags have been studied for signal amplification, such as enzymes, metal irons, ferrocene (Fc) and quantum dot [23,24]. While the effect of signal enhancement based on single electrochemical tag is limited, nanomaterials have been selected as supporting matrix to increase the loading of these tags and enhance the detection sensitivity [25,26]. Polystyrene sphere (PS), due to its facile synthesis condition, low cost and narrow size distribution have found extensive applications as building blocks for various nanostructures, as synthesis template, as vehicles for drug delivery and as supporting matrix for the immobilization of immunosensor signal tags [27–29]. For immunosensor application, the signal tags can be either

T. Li et al. / Sensors and Actuators B 186 (2013) 768–773

incorporated into the nanosphere or immobilized onto the nanosphere surface. In this work, we designed electrochemical label based on cadmium ion (Cd2+ ) or Fc incorporated PS (PS-Cd2+ , PS-Fc) for detection antibody, and developed an ultrasensitive multiplexed immunosensor for the simultaneous detection of interleukin-6 (IL6) and interleukin-17 (IL-17). Both of IL-6 and IL-17 are cytokines that secreted by T-cells and acted as mediators that stimulate immune-response [30–33]. The electrochemical tag incorporated PS were prepared simply with the addition of Cd2+ or Fc into the precursor solution that used to synthesize PS. After the immobilization of detection anti-IL-6 antibody and detection anti-IL-17 antibody (Ab2 ) onto PS-Cd2+ and PS-Fc, respectively, the resulting bioconjugates can selectively bind to their specific antigen target. The electrochemical current of the two signal tags can be easily distinguished due to the large separation of the two redox peak potentials, one at around −0.8 V and another at around +0.25 V. With the immobilization of the capture anti-IL-6 and antiIL-17 antibodies (Ab1 ) together onto one electrode, the resulting immunosensor displays good performance for simultaneous detection of IL-6 and IL-17 with high sensitivity, good reproducibility and negligible cross-reactivity. This proposed multiplex immunosensor platform can be easily extended to the detection of other protein biomarkers. 2. Experimental 2.1. Apparatus and reagents Interleukin-6 (IL-6), interleukin-17 (IL-17), human antirabbit IL-6 antibody and human anti-rabbit IL-17 antibody were obtained from Santa Cruz Biotechnology, Inc. (CA, USA). Poly(diallyldimethylammonium chloride) (PDDA, 20%, w/w in water, MW = 200 000–350 000) and chitosan were purchased from Sigma–Aldrich. Ferrocene, 2,2 -azobisisobutyronitrile (AIBN), styrene (St) and polyvinglpyrrolidone (PVP) were bought from Shanghai Sinopharm chemical reagent company (Shanghai, China). A 0.1 M phosphate buffer solution (PBS, pH 7.4) was used as supporting electrolyte during electrochemical measurements. All other reagents were of analytical grade and deionized water (MillQ, 18.2 M) was used throughout the study. All electrochemical measurements were performed on a CHI 650D electrochemical workstation (Shanghai CH Instruments Co., China). A conventional three-electrode system was used for all electrochemical measurements: a glassy carbon electrode (GC, 3 mm in diameter) as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. Scanning electron microscopic (SEM) images were obtained from Nova NanoSEM 230 (FEI, USA). Enzyme-linked immunosorbent assay (ELISA) of serum samples were performed by ELISA kit purchased from R&D Systems, Inc. (Minneapolis, USA). 2.2. Synthesis of Cd2+ or Fc incorporated PS For the synthesis of Cd2+ incorporated PS, 0.6 g of PVP, 19 mL of ethanol and 1 mL of water were mixed together and stirred under nitrogen atmosphere. After 30 min, temperature of the reaction was increased rapidly to 70 ◦ C. Then, 5 mL of St and 0.05 g of AIBN were mixed and added into the above reaction solution. After reaction of another 2 min, Cd(NO3 )2 was added into the reaction mixture to reach a concentration of about 0.7 mol/L. The reaction was continued for another 12 h at nitrogen atmosphere, and then cooled naturally. The synthesized PS was separated with centrifuge followed by washing with ethanol and water, and then dried to obtain white powder.

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To synthesize Fc incorporated PS, 0.6 g of PVP and 19 mL of ethanol were mixed together and stirred. After 30 min, temperature was increased to 70 ◦ C followed by the addition of 5 mL of St and 0.05 g of AIBN. After 2 min, Fc was added into the reaction mixture to reach a concentration of 10 mM. The reaction was continued for another 12 h at nitrogen atmosphere. The synthesized PS was also separated and washed extensively. 2.3. Preparation of PS-Cd2+ -Ab2 and PS-Fc-Ab2 The synthesized PS was dispersed into a 0.5% PDDA aqueous solution containing 0.5 M NaCl to reach a concentration of about 1 mg/mL and stirred for 1 h. The resulting PS was centrifuged and washed twice with water to obtain PDDA modified PS. Then, the PDDA modified PS was dispersed into 13 nm colloidal AuNPs and stirred for 30 min to adsorb AuNPs onto PS surface. Finally, the modified PS-Cd2+ was added into 0.1 mg/mL of detection anti-IL6 antibody solution, while the PS-Fc was added into 0.1 mg/mL of detection anti-IL-17 antibody solution. The mixture was gently mixed for 3 h and centrifuged. The obtained bioconjugate was stored at 4 ◦ C before use. 2.4. Preparation of the immunosensor To fabricate the immunosensor, Ab1 was immobilized onto graphene surface. First, the synthesized graphene was added into 1% chitosan (w/w) solution under stirring for 1 h. After centrifuge, the functionalized graphene was mixed with AuNPs solution. The mixture was stirred for another 1 h, and then AuNPs functionalized graphene was obtained after another round of centrifuge and extensive wash. For the adsorption of Ab1 onto AuNPs functionalized graphene, 1 mL of graphene solution (1 mg/mL) was mixed with 1 mL of Ab1 solution (containing 0.1 mg/mL of capture IL-6 antibody and 0.1 mg/mL of capture IL-17 antibody). The mixture was stirred gentle for 3 h and the free antibodies were separated by centrifuge. The Ab1 modified graphene (graphene-Ab1 ) was re-dispersed in PBS (1 mg/mL) and stored at 4 ◦ C before use. Onto the GC electrode, 5 ␮L of graphene-Ab1 buffer solution was added. The electrode was dried and washed with buffer. Then it was incubated in 1 wt% BSA solution for 30 min to eliminate nonspecific binding between the antigen and the electrode surface. Subsequently, buffer solution with different concentration of IL6 and IL-17 was added onto the electrode surface and incubated for 1 h at room temperature. After extensive wash, finally, mixture containing 1 mg/mL of PS-Cd2+ -Ab2 and PS-Fc-Ab2 was dropped onto the electrode surface and incubated for another 1 h. After another round of wash, the electrode was ready for measurement. To test the electrochemical signal of the immunosensor, 2 ␮L of tetrahydrofuran (THF) solution was dropped onto the electrode surface. After the evaporation of the THF, the electrode was washed gently and then a square wave voltammetry (SWV) scan from −1.2 to +0.8 V with a pulse amplitude of 25 mV, a pulse frequency of 15 Hz, and a quiet time of 2 s was performed to record the electrochemical responses for simultaneous and quantitative detection of IL-6 and IL-17. 3. Results and discussion 3.1. Characterization of the synthesized PS The synthesis of Cd2+ or Fc incorporated PS was simple, with the addition of Cd2+ or Fc into the precursor solution that used to synthesize PS, these electrochemical tags are incorporated into PS during the PS formation process. The synthesized PS-Cd2+ and PS-Fc were first characterized by SEM. As shown in Fig. 1, the PS display

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T. Li et al. / Sensors and Actuators B 186 (2013) 768–773

Fig. 1. SEM images of PS-Cd2+ and PS-Fc.

Cd(ox)

Cd(red)

Fc(ox)

Fc(red)

PS-Cd2+

PS-Fc

were significantly increased. For the PS-Cd2+ modified electrode, the current increased about 10 times compared to that without THF treatment. While for the PS-Fc, the peak current increased for about 15 times. These results indicated the large amount of electrochemical tags incorporated into the PS and the treatment of

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uniform morphology and narrow size distribution, indicating the procedure for the synthesis of the functionalized PS was quite efficient. The resulted PS-Cd2+ has diameter of about 300 nm, while the diameter of PS-Fc is around 1 ␮m. This result demonstrated the incorporation of these electrochemical tags into the PS did not destroy their structures. After the synthesis of electrochemical tag incorporated PS, the PS can be further used as labels in bioassay. Cd2+ and Fc are two of the most widely used signal tags in different kinds of bioassays due to their strong redox activity [34–36]. The current potential of Cd2+ and Fc are different and widely separated, so with the linking of two different antibodies onto PS-Cd2+ and PS-Fc, respectively, these two labels can bind to its specific antigen and produce two different redox current peaks (Scheme 1). To immobilize the antibodies onto the PS surface, a positively charged polyelectrolyte PDDA layer was first coated onto the PS for the following adsorption of negatively charged AuNPs. The immobilized AuNPs on the PS surface can then be utilized as anchor sites for Ab2 to prepare the labels. To detect the electrochemical tag contained in the PS, two different methods were tested. Solutions containing PS-Cd2+ or PS-Fc were dropped onto two electrode surface and then dried naturally. As shown in Fig. 2, for direct measuring the modified electrode, only a very small peak current was observed (curve a). This might be ascribed to the insulating properties of PS that prevented the detection of electrochemical tags contained in the PS. The small peak currents could mainly originated from the electrochemical tags adsorbed on the PS surface. In another method, onto the modified electrode, THF solution was added to dissolve the PS and release the contained electrochemical tags onto electrode surface. Through such method, the electrochemical peak currents

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Ab1 AuNPs Graphene GC Scheme 1. Schematic representation for the construction of the immunosensor.

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E vs SCE, V Fig. 2. (A) Square wave voltammetry (SWV) response of the PS-Cd2+ modified electrode without (a) and with (b) THF treatment. (B) Cyclic voltammetry (CV) response of the PS-Fc modified electrodes without (a) and with (b) THF treatment.

T. Li et al. / Sensors and Actuators B 186 (2013) 768–773

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Fig. 3. CV characterization of the immunosensor at each modification step in 5 mM Fe(CN)6 3− containing 0.1 M NaCl, (a) blank GC, (b) graphene-Ab1 modified GC, (c) after BSA blocking and the capture of 1 ng/mL of IL-6 and 1 ng/mL of IL-17, (d) after the capture of secondary antibody label.

PS with THF successfully released the tags onto electrode surface for the following detection.

3.2. Fabrication of the electrochemical immunosensor In this work, graphene was used for the simultaneous immobilization of capture anti-IL-6 and capture anti-IL-17 antibodies due to its large specific surface area and good conductivity. The large specific surface area of graphene will increase the loading of antibodies while its good conductivity can enhance the redox current of Fc and Cd2+ [37]. To immobilize the antibodies onto graphene, positively charged chitosan layer and negatively charged AuNPs were sequentially anchored onto graphene for the following adsorption of antibodies. With the immobilization of two antibodies onto electrode together and the binding of their specific antigen, PS-Cd2+ -Ab2 and PS-Fc-Ab2 can then selectively captured onto the electrode considering of the presence of IL-6 and IL-17 in the sample. The immunosensor preparation process was characterized by cyclic voltammetry (CV) in Fe(CN)6 3− solution. As seen from Fig. 3, compared to blank GC electrode, the immobilization of graphene-Ab1 onto electrode resulted in a significant increase of the peak current, which can be ascribed to the good conductivity of graphene. The following capture of antigen and the secondary antibody label caused the decrease of the peak current. These data indicated the successful modification of the immunosensor with each step. Fig. 4 shows the SWV of the immunosensor for the detection of mixture containing different concentration of IL-6 and IL-17. In the presence of 1 ng/mL of IL-6 and 1 ng/mL of IL-17, two current peaks were appeared at −0.8 V and +0.25 V, which reflected the identity of corresponding protein biomarkers (curve a). The current peak at −0.8 V is ascribed to Cd2+ due to the presence of IL-6 while the current peak at +0.25 V is ascribed to Fc due to presence of IL-17. In control experiment, when the sample contains only IL-6 or IL-17 (curve b and c), only one current peak exists, indicating negligible non specific reaction. Comparing curve a with curve b and c, it can be seen the current responses showed no obvious difference when the mixture contains one or two kinds of protein biomarkers. These results confirmed that the presence of one protein biomarker would not interfere with detection of the other, and the cross-reactivity between them could be negligible.

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E Vs SCE, V Fig. 4. SWV response of the immunosensor for the detection of different analyst; (a) 1 ng/mL of IL-6 and IL-17, (b) 1 ng/mL of IL-6, (c) 1 ng/mL of IL-17.

3.3. Analytical performance of the immunosensor Since we have demonstrated that two different protein biomarkers can be simultaneously detected by one single electrode, various concentrations of the two biomarkers were then tested by the immunosensor. Under optimized assay conditions, the peak currents were increased with the increasing concentration of biomarkers (Fig. 5A). The calibration curve shows good linear relationships between the peak currents and the logarithm of the biomarkers concentrations. For IL-6 (Fig. 5B), the linear range is from 1 pg/mL to 1 ng/mL with a correlation coefficient of 0.996. For IL-17 (Fig. 5C), the linear range is from 2 pg/mL to 1 ng/mL with a correlation coefficient of 0.998. The detection limits for IL-6 and IL-17 were 0.5 pg/mL and 1 pg/mL (S/N = 3), respectively. The wide linear range and low detection limits can be attributed to the significant loading of electrochemical tags into PS, which greatly amplified the current signals. In addition, the large surface of PS could increase the loading of Ab2 and enhance the opportunity of antibody–antigen interaction. The selectivity of the immunosensor was further investigated. Some proteins such as prostate specific antigen (PSA), human IgG, and tumor necrosis factor ␣ (TNF-␣) were used as the possible interferences to study the specificity of the proposed immunosensor. The response current of the immunosensor toward 0.1 ng/mL of IL-6 and 0.1 ng/mL of IL-17 were compared with that containing an interfere proteins of 1 ng/mL. The current variation due to the presence of interfere proteins are less than 7%, which demonstrated the proposed immunosensor displays high specificity and can be further used for detection of IL-6 and IL-17 in complicated serum samples. The reproducibility of the immunosensor was also studied. Five immunosensors were prepared in parallel and used for the detection of two samples. One sample contains 10 pg/mL of IL-6 and IL-17, while another samples contains 1 ng/mL of IL-6 and IL-17. The relative standard derivations (RSDs) of testing results for IL-6 and IL-17 were below 4.8% and 5.1%, which indicated the proposed immunosensor possess satisfactory precision and reproducibility. 3.4. Analysis of clinical serum samples To evaluate the potential application of the immunosensor, the immunosensor was utilized for the simultaneous detection of IL-6 and IL-17 in human serum samples (Samples are from The Second Xiang-ya Hospital). The serum sample were diluted by PBS and then

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T. Li et al. / Sensors and Actuators B 186 (2013) 768–773 Table 1 Detection of clinical serum samples using the proposed immunosensor and ELISA method.

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10 100 Concentration, pg/mL

1000

3

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Detection of IL-6 Immunosensor results (pg/mL) ELISA method (pg/mL) Relative deviation (%)

21.5 22.4 −4.0

35.5 35.1 1.1

47.2 46.3 1.9

49.3 48.3 2.0

Detection of IL-17 Immunosensor results (pg/mL) ELISA method (pg/mL) Relative deviation (%)

11.8 12.4 −5.1

15.5 15.9 −2.5

36.3 37.4 −2.9

49.3 46.7 5.5

4. Conclusions

Acknowledgements

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4 Current, µA

2

We are grateful for the support of National Natural Science Foundation of China (21105128, 21005047, 81200326), the Project of Shandong Province Higher Educational Science and Technology Program (J12LD17) and the Fundamental Research Funds for the Central Universities (2012QNZT150).

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References

3 2 1 0

1

In summary, we synthesized electrochemical tag (Fc and Cd2+ ) incorporated PS and applied the PS as label for multiplexed detection of IL-6 and IL-17. The detection of Fc or Cd2+ contained in the PS was achieved through voltammetry by first dissolve the PS with THF to release the electrochemical tags onto electrode surface. Due to the significant amount of electrochemical tags incorporated into PS, the sensitivity of the resulted immunosensor was significantly enhanced. The proposed electrochemical immunosensor shows acceptable reproducibility and accuracy for simultaneous detection of IL-6 and IL-17 with negligible cross-reactivity. When applying the immunosensor for serum sample analysis, satisfactory results were obtained. The method described here opens a new approach for simple, sensitive, and simultaneous determination of protein biomarkers, which shows great potential for accurate clinical disease screen.

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Concentration, pg/mL Fig. 5. (A) SWV response of the immunosensor for the simultaneous detection of different concentrations of IL-6 and IL-17. From (a) to (e), 5, 50, 100, 500, 1000 pg/mL. (B) Calibration curve of the immunosensor to different concentrations of IL-6. (C) Calibration curve of the immunosensor to different concentrations of IL-17.

determined by the immunosensor. The detection results were compared with results obtained by the commercial ELISA method. As shown in Table 1, the protein biomarker contents determined by the two methods agreed well with deviation below 6%, indicating feasibility of the proposed immunosensor for clinical applications.

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Biographies Ting Li, lecturer at the Second Xiang-ya Hospital, Central South University. His research interest includes cancer and transplantation biomarker detection and protein analysis. Bo Shu, lecturer at the Second Xiang-ya Hospital, Central South University. His research interest includes biomaterials and protein biomarkers. Bin Jiang, graduate student at the Second Xiang-ya Hospital, Central South University. His research interest includes biomaterials and drug delivery. Lu Ding, graduate student at College of Chemistry and Chemical Engineering, Qufu Normal University. Her research interest includes biosensors and electrochemistry. Haizhi Qi, professor at the Second Xiang-ya Hospital, Central South University. His research includes cancer biomarker detection and protein analysis. Minghui Yang, associate professor at College of Chemistry and Chemical Engineering, Central South University. He obtained his Ph.D. from Hunan University at 2007. His research interest includes biosensors, biomaterials and microfluidics. Fengli Qu, associate professor at College of Chemistry and Chemical Engineering, Qufu Normal University. She obtained her Ph.D. from Hunan University at 2008. Her current research interest includes biosensors and electrochemistry.