Ultrasensitive Multiplexed Immunoassay for Tumor Biomarkers Based

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Ultrasensitive Multiplexed Immunoassay for Tumor Biomarkers Based on DNA Hybridization Chain Reaction Amplifying Signal Jinjin Guo, Junchun Wang, Junqing Zhao, Zilin Guo, and Yuzhong Zhang* College of Chemistry and Materials Science, the Key Laboratory of Functional Molecular Solids, Ministry of Education, Anhui Key Laboratory of Chem-Biosensing, Anhui Normal University, Wuhu 241000, People’s Republic of China S Supporting Information *

ABSTRACT: In this work, a novel electrochemical immunoassay protocol has been reported for simultaneous determination of multiple tumor biomarkers based on DNA hybridization chain reaction (HCR) for signal amplification. Alpha-fetoprotein (AFP) and prostate specific antigen (PSA) were selected as model biomarkers. The immunoassay protocol contained primary antibodies immobilized on gold nanoparticles (Au NPs), secondary antibodies conjugated with DNA concatemer from HCR of primer, auxiliary probe, and signal probe labeled with signal molecules (methyleneblue (MB) and ferrocene (Fc)). In the presence of target biomarkers, the sandwich immunocomplex was formed between the primary antibodies and secondary antibodies bioconjugates carrying numerous signal molecules. As a result, two well-resolved reduction peaks, one was at −0.35 V (corresponding to MB) and other was at 0.33 V (corresponding to Fc; both vs SCE), were obtained in differential pulse voltammetry, and peak currents changed were related to the level of biomarkers. Under optimal conditions, the electrochemical immunoassay exhibited a wide linear response range (0.5 pg mL−1 to 50 ng mL−1) and low detection limits (PSA, 0.17 pg mL−1; AFP, 0.25 pg mL−1) (at S/N = 3). In addition, the immunoassay was evaluated by analyzing simulate human serum sample, and the recoveries obtained were within 99.4−107.6% for PSA and 97.9−108.2% for AFP, indicating the immnuoassay could be applied to the simultaneous detection of AFP and PSA in human serum samples. KEYWORDS: immunosensor, gold nanoparticles, hybridization chain reaction, ferrocene, methyleneblue, differential pulse voltammetry

1. INTRODUCTION Quantitative and sensitive detection of multiple biomarkers related to cancer is of great importance in screening and clinical diagnosis.1,2 The multiple immunoassay provided more accurate information for cancer diagnosis, up to now, different multiple immunoassays have been reported such as optical and electrochemical methods.3−7 Among these methods, electrochemistry-based multiple immunoassay has become one of the predominant analytical techniques due to simple instrumentation, high sensitivity, and selectivity.8,9 In immunoassay, signal amplification technologies have been employed for amplifying response signal to detect low level of biomarkers including enzyme,10,11 nanomaterials12−14 and molecular biotechnology.15−18 The traditional enzyme labeled antibodies often employed the ratio of 1:1 for enzyme and detection antibody, resulting in the low sensitivity of immunoassays. Enhancing immobilized amount of enzymes and antibodies was beneficial to improve the sensitivity of immunoassay, so, various nanomaterials were utilized in immunoassay. For example, silica nanoparticles, carbon nanospheres, and carbon nanotubes were employed to assemble enzyme and antibodies, and sensitivity has been improved obviously. Recently, DNA concatemer, a linear polymeric structure from continuous hybridization reaction of short DNA fragments, has caused considerable attention. In design, © XXXX American Chemical Society

different signal molecules can be selected to label short DNA fragments; as soon as the DNA concatemer was formed via HCR, numerous signal molecules were attached on the long concatemer. As a result, the electrochemical signal is amplified remarkably. Several ultrasensitive electrochemical DNA biosensors have been reported based on DNA concatemer amplifying signal.19,20 Tang’s group21 utilized HCR to amplify electrochemical signal and realized ultrasensitive detection of lgG (the detection limit is 0.1 fg mL −1 ). Therefore, immunoassy for multiple biomarkers based on nanomaterial and signal amplification from HCR has caused considerable attention. On the basis of our previous work and inspired from Tang’s work, we report here an ultrasensitive multiple immunoassay for simultaneous detection of AFP and PSA based on the amplification capability of HCR. In this study, two primary antibodies (Ab1) were immobilized on the surface of Au NPmodified glassy carbon electrode (GCE) and secondary antibodies (Ab2) labeled with primer were employed to hybridize with auxiliary probe and signal probe labeled with MB or Fc to form a long DNA concatemer. In the presence of Received: January 21, 2016 Accepted: March 2, 2016

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DOI: 10.1021/acsami.6b00756 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scanning electron microscopy (SEM, S-4800, Hitachi, Japan) was used to obtain the morphology of the Au NPs. 2.3. Preparation of Ab2 Labeled with Primer Bioconjugates (Ab2-S). The preparation of Ab2 labeled with the primer bioconjugates was carried out as follows: Briefly, 200 μL EDC (300 mM) and 200 μL NHS (100 mM) were first injected into 2.0 mL of anti-PSA (Ab2,PSA 1 mg mL−1) and reacted for 1 h. Afterward, the amino-labeled DNA primer (S0, 1.0 OD) was added into the solution above. After gently shaking for 15 min, the mixture was transferred to the refrigerator at 4 °C and reacted for overnight. During the process, the Ab2 was linked to DNA primer via the amidation of NH2 and COOH. Following that, the mixture was high-speed centrifuged for 25 min, and removed excess EDC and NHS. The bioconjugates obtained were redispersed in 1.0 mL PBS of pH 7.4 and stored in freezer for further use. Similarly, anti-AFP (Ab2,AFP)-S1 bioconjugates were synthesized according to the similar procedures of the Ab2,PSA-S0 above. 2.4. Fabrication of the Immunoassay Protocol. The immunoassay protocol is illustrated in Scheme 1. First, Au NPs were deposited on the surface of cleaned GCE (Notes: Au NPs/GCE) (the bare electrode was treated according to our previous reported22). The reason for depositing Au NPs is that Au NPs possess good conductivity and high surface areas which could improve the conductivity of the electrode interface and load amount of primary antibody (Ab1). Then, 10.0 μLAb1 (PSA, AFP)mixture was dropped on the surface of Au NPs and kept for 12 h in a refrigerator (Notes: Ab1/Au NPs/GCE). Next, it was incubated with 0.5% BSA solution to eliminate possible nonspecific sites, followed by rinsing with washing buffer for further use. The immunoassay contained three steps as follows: First, 300 μL of 2.0 μM auxiliary probe (H1 or H3) hybridized with 300 μL 2.0 μM signal probe (H2 or H4) in hybridization buffers for 3 h at 37 °C to form the DNA concatemer. Second, the electrode modified with PSA and AFP antibodies was incubated with the various concentrations of PSA and AFP antigens (or serum samples) for 45 min at 37 °C. Next, it was further incubated with Ab2-S bioconjugates for another 45 min and 1.0 μM DNA concatemer carrying multicopies MB and Fc for 75 min at 37 °C. Finally, DPV technique was employed to record the responsive signal in 5.0 mL PBS of pH 7.0 containing 0.1 M NaClO4 with the potential range from −0.7 to 0.5 V, pulse amplitudes of 0.05 V, pulse width of 0.08 s and sample width of 0.0167 s.

target proteins, the sandwich immuocomplex was formed, and the electrochemical signal was greatly increased because of the long concatemer carrying numerous signal molecules (Fc and MB). The peak difference was about 680 mV; therefore the cointerference from AFP and PSA was neglected, indicating the immunoassay can sensitive detect two biomarkes in one run and do not interfere each other. To the best of our knowledge, the electrochemical immunoassay for two biomarkers has not been reported based on HCR mechanism.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Reagents. Prostate specific antigen (PSA), PSA antibody (anti-PSA), alpha-fetoprotein antigen (AFP), AFP antibody (anti-AFP) and carcinoembryonic antigen (CEA) were purchased from Biocell Biotech. Co., Ltd. (Zhengzhou, China) and stored in refrigerator at 4 °C. Bovine serum albumin (BSA), Chloroauric acid (HAuCl4·4H2O), ascorbic acid (AA) and L-cysteine (L-cys) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). sodium perchlorate (NaClO4), N-Hydroxysuccinimide (NHS), N-(3-(Dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC), Dopamine (DA)were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade and used without further purification. All the sequences of oligonucleotides were from Shanghai Sangon Biological Co., Ltd. (Shanghai, China), and various DNA sequences were listed in Table 1.

Table 1. Various DNA Sequences Primer S0: 5′-NH2-(CH2)6-ATG-GTG-GTA-GTT-GGA-GCT-G-3′ S1: 5′-NH2-(CH2)6-CCT-AGA-TGG-ACG-AAG-TAG-A-3′ Auxiliary Probe H1: 5′-ACT-CTT-GCC-TAC-GCC-ACC-AGC-TCC-AAC-TAC-CAC-3′ H3: 5′-GTC-ACG-TAA-CGT-ATA-GAT-CTA-CTT-CGT-CCA-TCT-3′ Signal Probe H2: 5′-GTG-GCG-TAG-GCA-AGA-GTG-TGG-TAG-TTG-GAG-CTG(CH2)6-MB-3′ H4: 5′-TCT-ATA-CGT-TAC-GTG-ACA-GAT-GGA-CGA-AGT-AGA(CH2)6-Fc-3′

3. RESULTS AND DISCUSSION 3.1. Principle and Investigation of DNA Concatemer. In this work, antibodies labeled with primer (Ab2,PSA-S0; Ab2,AFP-S1) were prepared via amidation reaction. In auxiliary probes, 17 base from H1 or H3 at 5′ end are complementary with 17 base of signal probes H2 or H4 at 3′ end, and other 16 base of H1 or H3 are complementary with 16 base of H2 and H4.The auxiliary probes (H1, H3) hybridized with signal probes (H2 and H4)labeled at 3′ end with MB or Fc molecules to form long concatemer containing multicopies signal molecules.Thesandwich immunocomplex with long concatemer shown stronger electrochemical signal and two distinguishable electrochemical signals from Fc and MB were obtained. Peak intensities are related to level of biomarkers, therefore, the concentration of two biomarkers can be simultaneously detected by monitoring the change in signal intensities. As described above, the amplification of the electrochemical signal was implemented by the formation of the long concatemer between auxiliary probe and signal probe. To clarify the formation of DNA concatemer, we used gel electrophoresis to investigate the characteristics DNA concatemer. The mixture containing 2.5 μL DNA sample and 5 μL of loading buffer was dropped into the grooves and performed at a constant potential of 100 V for 40 min and the results are shown in Figure 1. Lanes 2 and 5 showed the gel electrophoresis images of H1 and H3 alone, two bright spots

a

Note: DNA sequences at 3′ end are complementary with other at 5′ end. Phosphate buffer saline (PBS) with various pH values were prepared by adjusting the ratio of the solution of 0.1 M Na2HPO4 and 0.1 M NaH2PO4. Various DNA sequences solutions were prepared with 0.01 M PBS (pH 7.4). Hybridization solution was 0.01 M PBS containing 0.1 M NaCl (pH 7.4), electrochemical test solution was 0.1 M PBS containing 0.1 M NaClO4 (pH 7.0) and washing buffer (0.1 M PBS containing 0.05% (w/v) Tween (pH 7.0)). The serum samples were from the clinical laboratory of the Yiji Shan Hospital (Wuhu, China). Twice-quartz-distilled water was used in this study. 2.2. Apparatus. Electrochemical measurements including differential pulse voltammetry (DPV), electrochemical impedance spectra (EIS) and cyclic voltammetry (CV) were performed on a CHI 650C electrochemical workstation (Shanghai Chenhua Instruments Co., Ltd., China). The three-electrode system consist of a glassy carbon electrode (GCE, 3.0 mm in diameter) or the modified electrode as working electrode, a platinum wire as auxiliary electrode, and a saturated calomel electrode (SCE) as reference electrode. Gel electrophoresis experiment was performed on a 1.0 wt % agarose gel containing 1.0 × trisacetic acid-EDTA (TAE) and 5 × loading buffer containing nucleic acid dye (Generay Biotech Co., Ltd., Shanghai, China). The gel images were photographed by Tanon 1600 gel imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China). B

DOI: 10.1021/acsami.6b00756 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. (A) Formation of the DNA Concatemer; (B) Schematic Representation of the Fabrication Procedure of the Immunosensor

respectively. The weakness spot at lane 3 might be signal probes labeled with MB were not migrated during electrophoresis. The phenomenon was similar to Tang’s reported.21 And lanes 4 and 7 appeared two long and wide bands, indicating that formation of DNA concatemer via hybridization chain reaction. 3.2. Characterization of the Immunoassay Fabrication. The characterization of Au NPs is shown in Figure S1. After electrodepositing for 20 s in1.0 mg mL−1 HAuCl4 solution at −0.20 V, it could be observed that Au NPs were distributed uniformly on the surface of GCE and did not aggregate, the diameter of Au NPs was estimated about 25 ± 2 nm. EIS technique was used to investigate the immunoassay fabrication and the results are shown in Figure 2A.The bare GCE showed a small semicircle (curve a), the electrode showed a straight line approximately (curve b) after Au NPs were electrodeposited, indicating that Au NPs have good conductivity. However, the semicircle of curves increased clearly (curves c and d) after Ab1 and BSA were immobilized on the surface of Au NPs, indicating that Ab1 and BSA are

Figure 1. Gel electrophoresis (lane 1, DNA marker; lane 2, 1 μM H1; lane 3, 1 μM H2; lane 4, 1 μM H1 + 1 μM H2; lane 5, 1 μM H3; lane 6, 1 μM H4; lane 7, 1 μM H3 + 1 μM H4).

were observed and the base number of H1 and H3 did not change, and lanes 3 and 6 showed the images of H2 and H4,

Figure 2. (A) EIS curves for the assembled step of the immunosensor in pH 7.0 PBS containing 5 mM [Fe(CN)6]3−/4−‑. Curves: (a) bare GCE, (b) Au NPs/GCE, (c) Ab1/Au NPs/GCE, (d) BSA/Ab1/Au NPs/GCE, (e) Ag/BSA/Ab1/Au NPs/GCE, (f) Ab2-S/Ag/BSA/Ab1/Au NPs/GCE. (B) CVs of the immunosensor in absence (black curve) and presence of DNA concatemer (red curve)in pH 7.0 PBS containing 0.1 M NaClO4. C

DOI: 10.1021/acsami.6b00756 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 3. Effects of experimental conditions on responsive signal: (A) pH values; (B) incubation time between Ag and Ab2-S; (C) incubation time between Ab2-S and DNA concatemer.

Figure 4. DPVs of the immunosensor when sandwich immunocomplex hybridized with auxiliary probe and signal probe in sequence (curve a); and DNA concatemer (curve b).

Figure 5. Cross-reactivity investigation of the immunosensor: (a) in absence of target antigen, (b) in the presence of 0.1 ng mL−1 PSA, (c) 0.1 ng mL−1 AFP, and (d) 0.1 ng mL−1 AFP + 0.1 ng mL−1 PSA in 0.1 M PBS (pH 7.0), respectively.

nonconductivity and hinder the electron transfer of [Fe(CN)6]3‑/4‑. After it was incubated with biomarker antigens (curve e) and Ab2-S bioconjugates (curve f), the semicircles were increased obviously due to they are no conductive and inhibit electron transfer of [Fe(CN)6]3‑/4‑. Finally, it was incubated with DNA concatemer, two distinguishable peaks of MB and Fc in CVs were observed in Figure 2B, indicating the sandwich immunocomplex formation carrying multicopies signal molecules. In addition, CV technique was also used for demonstrating the assembled process of the immunoassay. And the results are shown in Figure S2. Compared with bare electrode (curve a), the electrode modified with Au NPs showed higher peak

current, indicating Au NPs have excellent conductivity (curve b). When two different Ab1 were immobilized on the surface of Au NPs/GCE, the peak current decreased clearly (curve c). Subsequently, peak currents were decreased continuously (curves d and e) after it was blocked out with BSA and incubated with target biomarkers, respectively. These facts are in accordance with our predicted. 3.3. Optimization of Experimental Conditions. As is well-known, experimental conditions affected analytical performance of immunoassay. In this study, incubation and hybridization time and pH values were optimized. All results are shown in Figure 3. Protein activity and electrochemical signal of signal molecules are related to pH, from Figure 3A, it D

DOI: 10.1021/acsami.6b00756 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 6. (A) DPV responses of the immunosensor after incubation with different concentrations of target antigens from 0.5 pg mL−1 to 50 ng mL−1 in pH 7.0 PBS containing 0.1 M NaClO4. (B, C)Calibration curves between peak currents versus PSA, and AFP concentrations, respectively.

Table 2. Determination Results of Clinical Serum Samples Using the Immunoassay

1 2 3

added value PSA

found value AFP

average value PSA

RSD recovery AFP

serum samples PSA

AFP (ng mL−1)

PSA (ng mL−1)

AFP (ng mL−1)

PSA (%)

AFP (%)

0.05 0.50 1.50

0.50 1.50 10.0

0.054 ± 0.006 0.48 ± 0.08 1.59 ± 0.13

0.52 ± 0.06 1.61 ± 0.05 10.03 ± 0.12

0.052 0.497 1.614

0.504 1.633 9.79

0.4 2.12 3.07

2.52 3.23 5.89

104 99.4 107.6

100.8 108.2 97.9

response signal, the signal intensity was 2.1 fold higher than that in the absence of DNA concatemer (curve a),indicating the electrochemical signal was clearly amplified via HCR. 3.5. Cross-Reactivity of Immunoassay. The crossreactivity was evaluated by comparing the response signal in absence and presence of single and two biomarkers. And results are shown in Figure 5. No responsive signal was observed in absence of target biomarker (curve a), only one signal was obtained in the presence of single biomarker (curves b and c) and two distinguishable signals were obtained in the presence of two biomarkers (curve d). One was at −0.35 V (corresponding to MB) and other was at 0.33 V (corresponding to Fc), peak difference was about 680 mV, indicating that the detection of PSA and AFP would not cause any crossinterference each other. Thus, the immunoassay protocol could be used for simultaneous detection of two biomarkers. 3.6. Analytical Performance. Under the optimal conditions, the immunoassay method was employed to detect various concentrations of biomarkers antigen standard solution. And results are shown in Figure 6. It can be observed that two peak currents were increased gradually as the concentration of two biomarkers increased (see Figure 6A), and two calibration curves were obtained in the range from 0.5 pg mL−1 to 50 ng mL−1 for two biomarker antigens with the detection limit of 0.17 pgmL−1 for PSA and 0.25 pg mL−1 for AFP (S/N = 3) (see Figure 6B, C). For PSA, the linear regression equation was

could be observed that pH 7.0 PBS was suitable. Incubation time between Ab1 and Ag was selected 45 min according to our previous work and literature.23 The incubation time between Ag and Ab2-S conjugates was investigated. It could be observed from Figure 3B that peak currents both Fc and MB were increased rapidly as incubation time increased from 10 to 45 min. After that, it kept a stable value, indicating that the specific interaction of Ag and Ab 2 -S conjugates had reached equilibrium. Therefore, 45 min was selected in this study. Hybridization time between Ab2-S and long concatemer was also investigated, and shown in Figure 3C. It could be seen that peak currents both MB and Fc were increased as hybridization time from 15 to 75 min. After that, the currents increased slowly and tended to a stable. So, 75 min was selected in this study. 3.4. Comparison of Different Immunoassay Strategies. To investigate the signal amplification of the immunoHCR assay, we employed the Ab1/Au NP-modified electrode to detect 0.1 ng mL−1 AFP and PSA mixture using DPV. The results are shown in Figure 4.Two distinguishable weak peaks were observed (curve a) when the sandwich immunocomplex was hybridized with auxiliary probe and signal probe in sequence; and two stronger peaks were obtained (curve b) when sandwich immunocomplex was hybridized with long concatemer.The reason was ascribed to the concatemer carrying multicopies signal molecules which amplified the E

DOI: 10.1021/acsami.6b00756 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces I (μA) = 2.84 + 0.734C (ng mL−1) (R = 0.9962). For AFP, the linear regression equation was I (μA) = 2.53 + 0.714C (ng mL−1) (R = 0.9933). Compared with other multiplexed electrochemical immunoassay,23,25 the immunoassay exhibited a wider linear range and lower detection limit. 3.7. Specificity and Reproducibility. To evaluate the specificity of immunoassay, we selected exogenous substances including CEA, AA, DA and L-Cys to investigate the effect of the exogenous substance on electrochemical response signal in this study. The reason for the use of these substances was that they coexisted in serum sample and CEA was used to judge if the immunoassay had specificity. The comparative study was implemented by measuring the peak current changes before and after addition of the exogenous substances in low concentrations of biomarkers. The results obtained are shown with histogram in Figure S3. It could be observed that signal intensities were also kept a constant in the presence of large amount of interferences, indicating the immunoassay possessed good specificity.The reason for good specificity was ascribed to the specific interaction of Ab-Ag, and selected reductive signal from the signal molecules. Thus, AA interference can be removed, although DA has a reductive peak at 0.10 V, it do not affect the signal of signal molecules. Reproducibility was evaluated through the date from variation coefficient of intra-assay and the interassay, the intra-assay precision was evaluated by testing two standard mixture containing AFP and PSA in five times parallel. The variation coefficients of five times parallel test were 5.3% and 6.5% for 0.1 and 1 ng mL−1 AFP and 6.2% and 6.9% for 0.1, 1 ng mL−1 PSA, respectively. The interassay variation coefficients were 4.6% and 6.1% for 0.1 ng mL−1 AFP and PSA, respectively. Thus, the immunoassay showed a desirable reproducibility. 3.8. Application of the Immunoassay. To evaluate the applicability of the immunoassay in real samples, three samples including 0.05 ng mL−1 PSA and 0.50 ng mL−1 AFP, 0.50 ng mL−1 PSA and 1.50 ng mL−1 AFP, and 1.50 ng mL−1 PSA and 10.0 ng mL−1 AFP were prepared by spiking different concentrations of AFP and PSA standards into the serum specimens (the sample is from Yiji Shan Hospital of Wuhu, Anhui), standard addition method was utilized to obtain the content of biomarkers in serum. The results obtained were shown in Table 2. The recovery was within 97.9−108.2% for AFP and 99.4−107.6% for PSA (Seen Table 2), indicating the immnuoassay could be used for the detection of AFP and PSA in human serum samples in clinical diagnostics.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86 553 3869303. Fax: +86 553 3869303. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National NaturalScience Foundation of China(No. 20675002) and the Nature Science Foundation of Education Department of Anhui Province.

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REFERENCES

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4. CONCLUSIONS In summary, a ultrasensitive immunoassay protocol was developed for simultaneous detection of AFP and PSA based on HCR amplifying signal. MB and Fc were selected as responsive signal. The immunoassay exhibited wide linear response range and high sensitivity. In particular, the immunoassay provides a promising application in clinical analysis.

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The SEM characterization of Au NPs, CV of the immunoassay at the different assembled process, and the specificity of immunoassay (PDF)

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00756. F

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DOI: 10.1021/acsami.6b00756 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX