DNA-Based Hybridization Chain Reaction for Amplified

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DNA-Based Hybridization Chain Reaction for Amplified Bioelectronic Signal and Ultrasensitive Detection of Proteins Bing Zhang,† Bingqian Liu,† Dianping Tang,*,† Reinhard Niessner,‡ Guonan Chen,† and Dietmar Knopp‡ †

Key Laboratory of Analysis and Detection for Food Safety (Fujian Province & Ministry of Education of China), Department of Chemistry, Fuzhou University, Fuzhou 350108, P.R. China ‡ Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany S Supporting Information *

ABSTRACT: This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL−1. Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.

A

thermal cycling in an exponential way.13−15 Hence, thermal cycling methods are time-consuming, sometimes nonspecific, and limited to a thermostable enzyme and a laboratory setting. The rapidly emerging research field of the strand-displacement polymerase-based isothermal amplification provides excitingly possibilities for advanced development of new analytical tools and instrumentation for bioanalytical application.16,17 The protocol can be used for the continuous replication of one strand of a DNA duplex. The Willner group utilized rolling circle amplification (RCA) strategy for self-assembly of DNA nanotubes with controllable diameters.18 The Pugh group explored the impact of Phi29 multiple-stranddisplacement amplification on detection of large-scale copy number variants using oligonucleotide arrays.19 Unfortunately,

n ultrasensitive and feasible method for detecting and quantifying biomarkers is important in biological studies, clinical diagnostics, and treatment.1,2 Typically, the assay is performed using certain affinity ligands comprising aptamers and antibodies that specifically interact with the biomolecules and thus mediate a target-responsive signal transduction cascade.3,4 Recently, great attention has been focused on signal amplification without the use of enzymes, e.g., by employing DNA as amplified indicators.5−7 Existing DNA-based amplification techniques mainly include polymerase chain reaction (PCR) or ligase chain reaction (LCR)-based thermal cycling and strand-displacement polymerase-based isothermal cycling.8−10 PCR-based immunoassays have been reported for amplification of the signal after target recognition of capture antibody, whereas in the biobarcode assay and the DNAamplified electrochemical assay, it is preamplified using nanoparticles with a high ratio of DNA to capture antibody.11,12 The increase in the product amount was achieved by repeated © 2012 American Chemical Society

Received: April 3, 2012 Accepted: May 24, 2012 Published: May 24, 2012 5392

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Scheme 1. Immuno-HCR Assay Methoda

a

(a) Design and preparation of monoclonal mouse anti-human IgG-functionalized magnetic beads (Ab1-MBs) and polyclonal goat anti-human IgG/ initiator strands-conjugated gold nanoparticles (Ab2-S1-AuNPs). (b) Schematic depiction of the sandwiched immunoassays with the DNA-based hybridization chain reaction (MB: magnetic beads; AuNP: gold nanoparticle with 16 nm in diameter; S1: DNA initiator strand; Fc: ferrocenecarboxylic acid).

human serum (reagent grade, ≥95%, essentially salt-free, lyophilized powder) were purchased from Sigma-Aldrich (USA). Prostate specific antigen (PSA), cancer antigen 125 (CA 125), and dopamine (DA) were provided from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Monoclonal mouse anti-human PSA antibody (∼1.0 mg mL−1, clone 107−1A4, purified immunoglobulin) and polyclonal rabbit anti-human PSA antibody (conc. ∼1.0 mg mL−1, affinity isolated antibody, buffered aqueous solution) were obtained from Sigma-Aldrich. [Note: IgG samples are referred to human IgG unless otherwise stated.] Magnetic Fe3O4 beads (MBs, particle size: ∼100 nm) in an aqueous suspension with a concentration of 25 mg mL−1 were obtained from Chemicell GmbH (Berlin, Germany). Bovine serum albumin (BSA, 96%−99%), human serum albumin (HAS), and (3-glycidyloxypropyl) trimethoxysilane (C9H20O5Si, GOPS) were obtained from Sigma-Aldrich. Gold colloids with 16 nm in diameter were prepared and characterized as described.24 All other reagents were of analytical grade and were used without further purification. Ultrapure water obtained from a Millipore water purification system (≥18 MΩ, Milli-Q, Millipore) was used in all runs. Clinical serum samples were made available by Fujian Provincial Hospital, China. [For comparison, these specimens were assayed by Clinical Laboratory and Medical Diagnostics Laboratory, Fujian Provincial Hospital, Fuzhou 350001, China using electrochemiluminescence technique (Elecsys 2010, Roche, Switzerland).] The initiator strand (S1), H1*, and H2* were obtained from Sangon Biotech. Co., Ltd. (Shanghai, China). The sequences of oligonucleotides and ferrocene (Fc)-labeled probes are listed as follows:

RCA often depends on inefficient enzymes, and the steps required to prepare circular oligomer templates are timeconsuming and costly. In contrast, hybridization chain reaction (HCR) can also play the transduction role via an amplification approach.10,20,21 During this process, the single-stranded DNA (ssDNA) molecule is a versatile construction material that can be programmed to self-assemble into complex structures driven by the free energy of base pair formation without enzyme.22,23 Synthetic DNA machines can be powered by strand displacement interactions initiated by the sequential introduction of auxiliary DNA fuel strands.10 Typically, two stable species of DNA hairpins coexist in the solution until an initiator strand is introduced. The initiator triggers a cascade of hybridization events to yield nicked double helices analogous to alternating copolymers. Herein, we report the proof-of-concept of a novel and powerful immuno-HCR assay strategy for determination of larger target analytes, e.g., proteins, by coupling the amplification capability of the HCR with the sensitive electrochemical signal of ferrocene molecules conjugated to hairpin probes (Scheme 1). The assay protocol mainly involves (i) the formation of the sandwiched immunocomplex between the immobilized capture antibodies on the magnetic beads (Ab1-MBs) and the secondary antibodies on the gold nanoparticles conjugated with initiator strands (Ab2-S1AuNPs), (ii) the HCR reaction of DNA initiator strands on the Ab2-S1-AuNPs between H1* and H2* hairpin DNA molecules, and (iii) electrochemical measurement of magnetic immuno-HCR complexes with a sequential injection mode.

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EXPERIMENTAL SECTION

Materials and Reagents. Monoclonal mouse anti-human IgG (Fab specific) antibody (clone GG-6, designated as Ab1, ∼5.0 mg mL−1), polyclonal goat anti-human IgG (Fc specific) antibody (designated as Ab2, ∼2 mg mL−1), and IgG from 5393

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through a PTFE tubing with the flow cell. The ITO electrode was installed at the bottom of the cell, and an external permanent BaFe12O19 magnet with pot shape (10 mm in diameter and 5 mm in depth, 410−430 mT) was set under the ITO electrode. Ab1-MBs, Ab2-S1-AuNPs, H1* + H2* (DNA solution for hybridization chain reaction), and the washing/ detection solution (20 mM phosphate buffer containing 0.1 M NaClO4, pH 7.4) were introduced at 500 μL min−1 via a control valve-based injection loop, respectively. The target IgG was directly injected into the cell using a microsyringe. The assay consists of the following steps: (i) 200 μL of Ab1MBs (C[MB] ≈ 10 mg mL−1) was flowed into the detection cell and collected on the ITO surface with an external magnet; (ii) target IgG with different levels (100 μL) was injected into the detection cell and incubated for 30 min in the absence of external magnet to form the immunocomplexes on the MBs; (iii) the immunocomplexes were accumulated and washed in the presence of the external magnet; (iv) 200 μL of Ab2-S1AuNPs (C[Au] = 12 μM) was flowed into the cell and incubated for another 30 min in the absence of external magnet to construct the sandwich-type immunocomplexes; (v) the sandwiched immunocomplexes were accumulated and washed in the presence of the external magnet; (vi) 200 μL of H1* + H2* was added to the detection solution in the absence of external magnet and incubated for 80 min; and (vii) after washing, 20 mM phosphate buffer (PBS, pH 7.4) containing 0.1 M NaClO4 was flowed through the cell in the presence of the external magnet, and square wave voltammetry (SWV) from 550 to 150 mV (vs. Ag/AgCl) (amplitude: 25 mV; frequency: 15 Hz; increase E: 4 mV) was collected and registered as the sensor signals. The process of the immunoHCR assay with signal amplification is schematically represented in Scheme 1b. All incubations and measurements were performed using the stopped-flow technique at room temperature. After each step, the detection cell was washed with the external magnet using pH 7.4 PBS. Analyses were always made in triplicate. Elecsys 2010 Electrochemiluminescence Immunoassay (ECLIA) for Target IgG. To further elucidate the analytical reliability and applicable potential of the immunoHCR assay for testing of real samples, a commercially available ECLIA method was employed as a reference for comparison of the assayed results. The ECLIA assay is based on the property of the electrochemiluminescent label molecules relative to the tris(2,2′-bipyridyl)ruthenium(II)-tripropylamine system with a sandwich-type immunoassay format. The automated process consists of the aspiration of the sample, reagent, and microparticles, first incubation at 37 °C, additional reagent pipetting, secondary incubation at 37 °C, reaction mixture aspiration, and measurement. Initially, 50 μL of sample, a biotinylated monoclonal IgG-specific antibody, and a second monoclonal IgG-specific antibody labeled with a ruthenium complex were mixed and incubated for 9 min at 37 °C to form a sandwiched complex in the presence of IgG molecules. Following that, streptavidin-coated paramagnetic microparticles were added and the reaction mixture was incubated for another 9 min, during which the sandwich complex bound to the particles by streptavidin−biotin interaction. Afterward, the reaction mixture was transferred into the measuring cell for electrochemiluminescence measurement. Results were determined via an instrument-specific calibration curve and a master curve provided via the reagent barcode.

The design of the hairpin probes was adapted from the literature.8,10 In the hairpin sequences, loops are italicized and sticky ends are underlined. Preparation of Ab1-Conjugated Magnetic Beads (Ab1MBs). The antibody-conjugated magnetic beads were prepared and characterized as described in our previous report.25 Briefly, magnetic beads were initially separated using an external magnet and dried in the vacuum at 80 °C for 1 h. Then, 250 mg of MBs was added into 5.0 mL of 5% GOPS (v/v) in dry toluene and stirred with 500 rpm for 12 h at room temperature (RT). With the aid of an external magnet, the GOPSfunctionalized magnetic beads were separated and purified. Afterward, the synthesized MBs were washed 3 times with toluene and ethanol solution, respectively. The purified MBs were dried and activated in an oven at 80 °C for 1 h under a nitrogen atmosphere. Following that, 100 μL of monoclonal mouse anti-human IgG antibody (1.0 mg mL−1) was injected into the functionalized Fe3O4 aqueous solution with the concentration of 25 mg mL−1. The mixture was slightly stirred for 12 h at 4 °C to conjugate existing amino groups of Ab1 antibodies to epoxy groups of GOPS. The obtained Ab1-MBs were incubated with 10 mg mL−1 BSA-PBS at 37 °C for 1 h to block the unreacted and nonspecific sites. Finally, the asprepared Ab1-MBs were stored in 20 mM phosphate buffer (PBS, pH 7.4, 0.1 wt % sodium azide) with a final concentration of 25 mg mL−1 at 4 °C when not in use. The conjugation process of the Ab1-MBs is schematically illustrated in Scheme 1a (left) and Scheme S1 of the Supporting Information. Preparation of DNA Initiator Strands (S1) and Ab2Conjugated Gold Nanoparticles (Ab2-S1-AuNPs). The Ab2-S1-AuNP nanocomplexes were synthesized and prepared according to the literature with a little modification,6,12,26 as illustrated in Scheme 1a (right). Prior to experiment, 5 mL of 16 nm gold colloids (AuNPs, C[Au] = 24 μM) was adjusted to pH 9.0−9.5 by directly using 0.1 M Na2CO3 aqueous solution. Then, 200 μL of polyclonal goat anti-human IgG antibody (Ab2, 2 mg mL−1) was added into gold colloids and incubated for 20 min at room temperature. During this process, Ab2 antibodies were covalently bound to gold nanoparticles via the dative binding between gold nanoparticles and free −SH groups of the antibody.27 Afterward, the alkylthiol-capped barcode DNA initiator strands (S1, 0.5 OD, see Figure S1 in the Supporting Information) was injected into the mixture. After gently shaking for 5 min, the mixture was transferred to the refrigerator at 4 °C for further reaction (overnight). Following that, the mixture was centrifuged (14 000g) for 25 min at RT. The pellet (i.e., S1/Ab2-functionalized gold nanoparticles, designated as Ab2-S1-AuNPs) was resuspended in 1.0 mL of 2 mM sodium carbonate solution (C[Au] = 120 μM) containing 1.0 wt % BSA and 0.1% sodium azide, pH 7.4, and stored at 4 °C until use. Flow-Through Electrochemical Immuno-HCR Assay Protocol. The electrochemical measurement of the immunoHCR assay method was carried out by coupling the CHI 630D Electrochemical Analyzer (CH Instruments Inc., Shanghai, China) with a flow-through detection cell using an Indium Tin Oxide (ITO, 5 wt % In2O3 + SnO2) working electrode, a platinum wire auxiliary electrode, and a saturated Ag/AgCl reference electrode. The flow-through detection setup is schematically illustrated in Scheme S2 (see the Supporting Information). The flow-through system consisted of a six-way valve equipped with a 1 mL syringe pump and connected 5394

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Figure 1. (a) TEM image of Ab2-S1-AuNPs after incubation with H1* and H2* and (b) gel electrophoresis (lane 1: DNA ladder; lane 2: 0.5 μM H1*; lane 3: 0.5 μM H2*; lane 4: 0.5 μM H1* + 0.5 μM H2*; lane 5: 0.5 μM H1* + 0.5 μM H2* + 1 mg mL−1 Ab2-S1-AuNP; lane 6: 0.5 μM H1* + 0.5 μM H2* + 3 mg mL−1 Ab2-S1-AuNP; lane 7: 0.5 μM H1* + 0.5 μM H2* + 6 mg mL−1 Ab2-S1-AuNP).

Figure 2. UV−vis absorption spectra of (a) Ab2-S1-AuNPs (1.0 mg mL−1) and (b) the same amount of Ab2-S1-AuNP samples after incubation with various concentrations of H1* + H2* for 30 min at room temperature. During the experiment “b”, the concentration of the Ab2-S1-AuNPs was 3 mg mL−1, while the concentrations of H1* + H2* were 0, 0.1, 0.2, 0.3, 0.4, and 0.5 μM from bottom to top, respectively. In this experiment, six samples were prepared in parallel. After incubation, these samples were centrifuged and dispersed into six 300 μL of distilled water, respectively, and finally evaluated using UV−vis absorption spectroscopy.

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RESULTS AND DISCUSSION Principle and Characteristics of Immuno-HCR Assay. In this work, the Ab1 -MBs were prepared by direct immobilization of monoclonal mouse anti-human IgG on the magnetic beads through the epoxy-amine reaction. Gold nanoparticles (16 nm) heavily functionalized with the DNA initiator strands and polyclonal goat anti-human IgG were utilized as secondary antibodies. The DNA hairpins H1* and H2* are dual-labeled with ferrocene moieties at each end. Each hairpin has a stem of 18 base pairs enclosing a hexanucleotide loop. Each also has an additional hexanucleotide sticky end at the 5′ end of H1* (complementary to the loop of H2*) and at the 3′ end of H2* (complementary to the loop of H1*). In a typical target IgG detection experiment, Ab1-MBs and Ab2-S1AuNPs initially sandwich the target IgG, generating a complex with a large ratio of the initiator strand (S1) and target IgG. The initiator strands on the Ab2-S1-AuNPs pair with the sticky end of H1*, which undergoes an unbiased strand-displacement interaction to open the hairpin. The newly exposed sticky end of H1* nucleates at the sticky end of H2* and opens the hairpin to expose a sticky end on H2*. This sticky end is identical in sequence to the initiator strands. In this way, each

initiator strand on the Ab2-S1-AuNPs propagates a chain reaction of hybridization events between alternating H1* and H2* hairpins to form a nicked double-helix. In this case, a ferrocene moiety on one probe is brought into close proximity to a ferrocene moiety on the neighboring probe. Thus, numerous ferrocene molecules are formed, each of which produces an electrochemical signal within the applied potentials. By monitoring the change of the electrochemical signal, we can indirectly determine the concentration of human IgG with high sensitivity. Importantly, in the absence of the initiator strands, both hairpin DNA molecules (H1* and H2*) are in the closed form and can not be conjugated onto the Ab1MBs. As described above, the amplification of the electrochemical signal was implemented by the formation of the long nicked DNA polymers between H1* and H2* on the Ab2-S1-AuNPs. To realize our design, we first used transmission electron microscopy (TEM, H-7650, Hitachi Instruments, Japan) and gel electrophoresis to characterize the Ab2-S1-AuNPs after incubation with H1* + H2*. As seen from Figure 1a, many long nicked DNA poly strands were observed on the AuNPs, indicating that the initiator strands on the Ab2-S1-AuNPs could 5395

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trigger a chain reaction of hybridization events between H1* and H2*. In contrast, the TEM image of pure Ab2-S1-AuNPs was almost the same as gold nanoparticles, and there were no flagellum-like structures on the gold nanoparticles (Figure S2 in the Supporting Information). The results revealed that the long nicked DNA poly strands could be formed in the presence of H1* and H2* with the guidance of the initiator strands on the AuNPs. This phenomenon could be also observed in the gel electrophoresis (Figure 1b). As seen from lanes 2 and 3, the base number of H1* and H2* oligonucleotides were almost the same, which was in agreement with our design. Significantly, the mixture of 0.5 μM H1* and 0.5 μM H2* did not cause their self-hybridization reaction (lane 4). When the Ab2-S1-AuNPs (1.0 mg mL−1) were added into the H1* + H2* mixture, H1* and H2* were partly consumed as a result of the HCR reaction (lane 5). The weakness of the spots at lane 5 might be the fact that the formed long-nicked DNA polymers with the Ab2-S1AuNPs were not migrated during the gel electrophoresis. With the increasing Ab2-S1-AuNPs, the HCR reaction was further carried out (lanes 6 and 7). Higher concentrations of the Ab2S1-AuNPs resulted in the increased consumption of H1* and H2*. Finally, the spots disappeared when 6 mg mL−1 of Ab2S1-AuNPs were injected into the mixture containing 0.5 μM H1* and 0.5 μM H2* (lane 7). By the same token, we also investigated the characteristics of the as-synthesized Ab2-S1AuNPs before and after incubation with H1* + H2* at various concentrations using UV−vis absorption spectroscopy (UV 1102, Techcomp, China). As seen from Figure 2a, there were two absorption peaks at 260 and 518 nm for the prepared Ab2S1-AuNPs, which were attributed to DNA molecules and gold nanoparticles, respectively. However, we did not observe the absorption peak of Ab2 antibodies. The reason might be the fact that the amount of the immobilized antibodies on the gold nanoparticles was relatively less. Importantly, it can be seen that the absorption peaks at 260 nm increased with the increasing concentration of H1* and H2* (Figure 2b), which was as a consequence of S1 hybridization chain reaction with H1* and H2*. In contrast, the characteristic peak for gold colloids at ∼518 nm did not change with the increase of H1* and H2* concentrations (Figure 2b). These results revealed that the HCR reaction could be progressed in the simultaneous presence of the Ab2-S1-AuNPs/H1*/H2*, thereby providing a precondition for immuno-HCR electrochemical assay. Comparison of Different Immunosensing Strategies. To investigate the signal amplification of the immuno-HCR assay during the electrochemical measurements, the asprepared Ab1-MBs were utilized for determination of 10 ng mL−1 IgG under the different conditions using square wave voltammetry (SWV) (Figure 3). No peak was observed when the Ab1-MBs were reacted with IgG and excess Ab2-S1-AuNPs (curve “a” in Figure 3). When the formed Ab1-MB-IgG-Ab2-S1AuNPs were incubated with H1* probes, an obvious square wave voltammetric (SWV) peak was acquired (curve “b” in Figure 3). However, the peak current was lower than that after reincubation with H2* (curve “c” in Figure 3). (Note: The process f rom curve “a” to curve “c” was designated as the 1:1 hybridization.) More inspiringly, when the incubation solution simultaneously contained H1* and H2*, the peak current heavily increased (curve “d” in Figure 3). This is most likely a consequence of the fact that the initiator strands on the Ab2-S1AuNPs could trigger the HCR reaction between H1* and H2* to form long nicked DNA polymers after “n” cycles. Moreover, the amplified signal using H1* + H2* (“n” cycles) was 407% of

Figure 3. SWV response curves of (a) Ab2-S1-AuNP-IgG-Ab1-MBs, (b) probe “a” after incubation with H1*, (c) probe “b” after incubation with H2*, and (d) probe “a” after simultaneous incubation with H1* + H2*. Potential scanning was from 550 to 150 mV (vs. Ag/AgCl; amplitude: 25 mV; frequency: 15 Hz; increase E: 4 mV). Measurements were performed in 20 mM phosphate buffer (pH 7.4) containing 0.1 M NaClO4.

H1* alone and 268% of 1:1 hybridization process, respectively. (Note: Herein, we also investigated the maximum number of hybridization cycles (n) via alternately incubation with H1* and H2* using 0.1 fg mL−1 target IgG as an example. Experimental results indicated that the eight hybridization cycles could be obtained. See Figure S3 in the Supporting Information.) For comparison, we also investigated the electrochemical response of the Ab1-MBs toward zero analyte. The peak currents were almost not changed, suggesting that H1* and H2* could not be conjugated on the Ab1-MBs in the absence of Ab2-S1-AuNPs. The results indicated that the immuno-HCR assay protocol could be utilized for detection of target IgG and the amplification of the electrochemical signal. Kinetic Characteristics and Optimization of the Immuno-HCR Assay. The question to be answered is whether the DNA concatamers on the AuNPs could be progressed with the increasing incubation time between the initiator strands and H1* + H2*. To clarify this point, the kinetic behaviors of the formed MB-Ab1-IgG-Ab2-S1-AuNPs were studied by monitoring the currents as a function of incubation time with H1* and H1* + H2*, respectively (10 ng mL−1 IgG used in this case). The peak currents increased with the time aged (Figure 4A). Compared with H1* + H2* (curve “b” in Figure 4A), H1* reaction with the initiator strands on the Ab2-S1-AuNPs (curve “a” in Figure 4A) was relatively fast at room temperature. The reason might be the fact that it took longer time for the S1 initiator strands on the MB-Ab1-IgGAb2-S1-AuNPs to open two hairpin structures (H1* + H2*) than that of one hairpin structure (H1*). Moreover, the assay using H1* + H2* exhibited higher current change than that of the use of H1* alone. Thus, 80 min was selected for the HCR reaction between S1 and H1* + H2*. Usually, the antigen−antibody reaction is adequately carried out at human normal body temperature (37 °C). Considering the possible application of the proposed immunoassay in the future, we selected room temperature (25 ± 1.0 °C) for the antigen−antibody interaction throughout the experiment. At this condition, we also investigated the effect of incubation time for the antigen−antibody reaction on the electrochemical signal of the immuno-HCR assays from 5 to 45 min using 1.0 pg mL−1 target IgG as an example (Figure 4B). To avoid confusion, the incubation times of the Ab1-MBs with target IgG were paralleled with those of the target IgG-Ab1-MBs with 5396

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Figure 4. (A) Electrochemical responses of Ab2-S1-AuNP-IgG-Ab1-MBs after incubation with (a) H1* and (b) H1* + H2* as a function of time, respectively. (B) Signal dependence of the immuno-HCR electrochemical assays on incubation time for the antigen−antibody reaction using 1.0 pg mL−1 target IgG as an example.

Figure 5. The interfering effects of sample matrix components on the electrochemical signal of the immuno-HCR assays. Initially, the human serum sample used for spiking was assayed using the immuno-HCR assay method, and various sample matrixes were then spiked into the human serum. Following that, the resulting mixtures were determined using the same method. Note: Using the spiking level for each interfering component was near the maximum concentration of the normal human serum in this case.

immuno-HCR assays, we challenged the system with several possible components in human serum, such as K+, Ca2+, Cl−, HCO3−, glucose (Glu), uric acid (UA), dopamine (DA), human IgG, and cancer antigen 125 (CA125). The reason for the use of these samples is that they usually coexist in the normal human serum. Close to the highest level for each sample (interfering matrix) in the normal human serum was studied as an example (inset of Figure 5a). These samples were assayed by spiking them into normal human serum sample, respectively. The comparative study was carried out by measuring the low concentration of target IgG and high concentration of interfering components based on the change in the current before and after addition of the interfering reagents. As indicated from Figure 5, higher current was observed with the target IgG than those of other components. These results clearly demonstrated the high specificity of the developed immuno-HCR assays. The precision and reproducibility of the immuno-HCR assays were evaluated using the variation coefficient (CV) of the intra- and interassay (CV, n = 3). The experimental results indicated that the CVs of the assays using the Ab2-S1-AuNPs and Ab1-MBs with the same batch were 4.6%, 7.3%, and 5.7% at the 10 fg mL−1, 10 pg mL−1, and 10 ng mL−1 IgG levels, respectively; the batch-to-batch reproducibility with various batches was monitored, and the CVs were 9.4%, 8.7%, and 10.3% at the above-mentioned levels, respectively. Hence, the precision and reproducibility of the immuno-HCR assays was acceptable.

Ab2-S1-AuNPs. As shown in Figure 4B, the currents increased with the increment of incubation time and tended to level off after 30 min. Hence, an incubation time of 30 min was selected for sensitive determination of IgG at acceptable throughput. Analytical Performance of the Electrochemical Immuno-HCR Assay. Under optimal conditions, the sensitivity and dynamic range of the electrochemical immuno-HCR assay were evaluated toward target IgG standards in 20 mM PBS, pH 7.4, containing 0.1 M NaClO4 by coupling DNA-based hybridization chain reaction with a sandwich-type immunoassay format. Figure S4 in the Supporting Information shows the electrochemical responses and dynamic range of the immunoHCR assays toward target IgG standards with various concentrations. As shown from Figure S4-a (Supporting Information), the SWV peak currents increased with the increasing of IgG concentration. The calibration plots displayed a good linear relationship between the peak currents and the logarithm of IgG concentrations in the range from 0.1 fg mL−1 to 100 ng mL−1 with a low detection limit of 0.1 fg mL−1 IgG estimated at a signal-to-noise ratio of 3σ (Figure S4-b in the Supporting Information). In contrast, the LOD was 1.0 ng mL−1 target IgG using H1* alone (Figure S5 in the Supporting Information). Although the system has not yet been optimized for maximum efficiency, the assay sensitivity of the use of H1* + H2* was 6 orders of magnitude lower than that of the use of H1* alone. To investigate the interfering effects of sample matrix components on the responses of the electrochemical 5397

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To illustrate the generality of our design, we applied this strategy to the detection of prostate-specific antigen (PSA) using the corresponding antibodies (see the Supporting Information for experimental details). The Ab2-S1-AuNPs were prepared using anti-PSA and the initiator ssDNA with gold nanoparticles. The immuno-HCR assays could detect PSA with a detection limit of 50 fg mL−1 (Figure S6 in the Supporting Information), which was 100-fold and 10-fold lower than those of magnetic separation immunoassay4 and nanoparticle-based biobarcodes,7 respectively.

In addition, the stability of Ab1-MBs and the Ab2-S1-AuNPs was also studied over a six-week period. When Ab1-MBs and the Ab2-S1-AuNPs were stored at 4 °C and measured intermittently (every 3−5 days), they retained 97.2%, 97.6%, and 77.3% (n = 3) of the initial signal after being stored for 2, 4, and 6 weeks, respectively. We speculate that the slow decrease of the signals was mainly attributed to the gradual deactivation of the immobilized biomolecules on the nanoparticles. Analysis of Real Samples and Evaluation of Method Trueness. The technique was further validated using assays for 15 clinical serum specimens with reference to a commercialized available electrochemiluminescence immunoassay (ECLIA) method. The obtained levels of IgG in these samples by the immuno-HCR assays displayed the deviations ranging from 1.8% to 9.1% with the referenced ECLIA method (Table 1).

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CONCLUSIONS In summary, we have developed a simple and highly sensitive amplified immunoassay method by coupling the DNA-based hybridization chain reaction with the biobarcode assay strategy. The signal could be amplified by the HCR-based reaction with hairpin structures in the presence of the initiator strands. Compared with other strategies, the method is sensitive and simple without the participation of enzyme molecules, thereby representing an excellent isothermal signal-amplification strategy. Significantly, the assay approach does not require sophisticated fabrication and is well suited for high-throughput biomedical sensing and application in both clinical and biodefense areas by controlling the target antibody.

Table 1. Comparison of the Assay Results for Clinical Serum Specimens Using the HCR-Based Immunoassays Herein Proposed and the Referenced ECLIA Method method; concentration (mean ± SD, n = 3, ng mL−1) sample no. 1 2 3 4 5 6a 7 8 9 10a 11 12 13 14 15

immuno-HCR assay 78.2 43.5 37.2 28.7 89.2 145.6 23.2 15.2 56.4 356.2 87.3 45.2 25.6 28.9 35.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.1 3.2 1.9 1.7 4.3 6.7 1.2 0.9 2.7 5.8 3.4 4.3 1.5 1.9 2.7

ECLIA 80.2 41.3 33.1 31.2 93.2 159.1 24.1 14.3 53.8 345.6 84.3 48.6 29.1 25.9 37.2

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.5 3.6 3.1 0.9 2.7 5.9 0.5 0.5 1.3 4.2 1.8 1.2 0.8 0.9 1.1

RSD (%)

texp

1.8 3.7 8.3 5.9 3.1 6.3 2.7 4.3 3.4 4.4 2.5 5.2 9.1 7.8 3.9

1.34 0.79 1.95 2.25 1.36 2.62 1.20 1.51 1.50 2.56 1.35 1.32 0.28 2.47 1.19

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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a

Samples 6 and 10 were initially diluted 10-fold with pH 7.4 PBS and then assayed by the immuno-HCR assay method.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (41176079, 41076059, 21075019), the National “973” Basic Research Program of China (2010CB732403), the Doctoral Program of Higher Education of China (20103514120003), the National Science Foundation of Fujian Province (2011J06003), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1116), and the Alexander von HumboldtFoundation of Germany.

Meanwhile, we also investigated the effect of various dilution ratios with different dilution chemicals on the analytical properties of the immuno-HCR assay (Table S1 in the Supporting Information). Statistical comparison of these results between two methods was performed using a t-test for comparison of means proceeded by the application of an Ftest. No significant differences were encountered between the two methods at the 0.05 significance level because the texp were in all cases below tcrit (tcrit[4, 0.05] = 2.77). To further embody the merits of the immuno-HCR assay for determination of IgG with ultralow concentrations, five samples including 0.5 fg mL−1, 5.0 fg mL−1, 100 fg mL−1, 1.0 pg mL−1, and 100 pg mL−1 target IgG were prepared by spiking IgG standards into new born calf serum, respectively. The contents measured by the immuno-HCR assays were 0.6 fg mL−1, 4.3 fg mL−1, 121.2 fg mL−1, 1.16 pg mL−1, and 98.3 pg mL−1 for the above five analyte concentrations, respectively. The recovery was 86−122%. These results demonstrated that our strategy provided assay performance comparable to the commonly used method and could be considered as an optional scheme for detection of target IgG in clinical diagnostics.

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REFERENCES

(1) Wang, F.; Elbaz, J.; Orbach, R.; Magen, N.; Willner, I. J. Am. Chem. Soc. 2011, 133, 17149−17151. (2) Wu, X.; Liu, H.; Liu, J.; Haley, N.; Treadway, J.; Larson, J.; Ge, N.; Peale, F.; Bruchez, M. Nat. Biotechnol. 2003, 21, 41−46. (3) Tabkman, S.; Lau, L.; Robinson, J.; Price, J.; Sherlock, S.; Wang, H.; Zhang, B.; Chen, Z.; Tangsombatvisit, S.; Jarrell, J.; Utz, P.; Dai, H. Nat. Commun. 2011, 2, 466 ; DOI: DOI: 10.1038/ncomms1477. (4) Nie, H.; Liu, S.; Yu, R.; Jiang, J. Angew. Chem., Int. Ed. 2009, 48, 9862−9866. (5) Brackmann, S. Angew. Chem., Int. Ed. 2004, 43, 5730−5734. (6) Nam, J.; Thaxton, C.; Mirkin, C. Science 2003, 301, 1884−1886. (7) Das, J.; Aziz, M.; Yang, H. J. Am. Chem. Soc. 2006, 128, 16022− 16023. 5398

dx.doi.org/10.1021/ac3009065 | Anal. Chem. 2012, 84, 5392−5399

Analytical Chemistry

Article

(8) Huang, J.; Wu, Y.; Chen, Y.; Zhu, Z.; Yang, X.; Yang, C.; Wang, K.; Tan, W. Angew. Chem., Int. Ed. 2011, 50, 401−404. (9) Saiki, R.; Gelfand, D.; Stoffel, S.; Scharf, S.; Higuchi, R.; Horn, G.; Mullis, K.; Erlich, A. Science 1988, 239, 487−491. (10) Dirks, R.; Pierce, N. Proc. Natl. Acad. Sci. 2004, 101, 15275− 15278. (11) Mason, J.; Xu, L.; Sheng, Z.; O’Leary, T. Nat. Biotechnol. 2006, 24, 555−557. (12) Wang, J.; Liu, G.; Munge, B.; Lin, L.; Zhu, Q. Angew. Chem., Int. Ed. 2004, 43, 2158−2161. (13) Towner, K.; Talbot, D.; Curran, R.; Webster, C.; Humphreys, H. J. Med. Microbiol. 1998, 47, 607−613. (14) Jonas, D.; Speck, M.; Daschner, F.; Grundmann, H. J. Clin. Microbiol. 2002, 40, 1821−1823. (15) van Beek, J.; zur Kausen, A.; Kranenbarg, E.; Warring, R.; Bloemena, E.; Craanen, M.; van de Velde, C.; Middeldorp, J.; Meijer, C.; van den Brule, A. Mod. Pathol. 2002, 15, 870−877. (16) Guo, Q.; Yang, X.; Wang, K.; Tan, W.; Li, W.; Tang, H.; Li, H. Nucl. Acid Res. 2009, 37, art. no. e20. (17) Gill, P.; Ghaemi, A. Nucleosides, Nucleotides Nucleic Acids 2008, 27, 224−243. (18) Wilner, O.; Orbach, R.; Henning, A.; Teller, C.; Yehezkeli, O.; Mertig, M.; Harries, D.; Willner, I. Nat. Commun. 2011, 2, art. no. 540. (19) Pugh, T.; Delarney, A.; Farnoud, N.; Griffith, S.; Li, M.; Li, H.; Qian, H.; Farinha, P.; Gascoyne, R.; Marra, M. Nucl. Acid Res. 2008, 36, art. no. e80. (20) Niu, S.; Jiang, Y.; Zhang, S. Chem. Commun. 2010, 46, 3089− 3091. (21) Choi, H.; Chang, J.; Trinh le, A.; Padilla, J.; Fraser, S.; Pierce, N. Nat. Biotechnol. 2010, 28, 1208−1212. (22) Peng, Y.; Choi, H.; Calvert, C.; Pierce, N. Nature 2008, 451, 318−322. (23) Venkataraman, S.; Dirks, R.; Rothemund, P.; Windfree, E.; Pierce, N. Nat. Nanotechnol. 2007, 2, 490−494. (24) Yuan, R.; Tang, D.; Chai, Y.; Zhong, X.; Liu, Y.; Dai, J. Langmuir 2004, 20, 7240−7245. (25) Tang, D.; Su, B.; Tang, J.; Ren, J.; Chen, G. Anal. Chem. 2010, 82, 1572−1534. (26) Hill, H.; Mirkin, C. Nat. Protoc. 2006, 1, 324−336. (27) Hermanson, G. Bioconjugate Techniques, 2nd ed.; San Diego: Academic Press, 2008.

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