Analytical Biochemistry 453 (2014) 16–21

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Dual signal amplification of surface plasmon resonance imaging for sensitive immunoassay of tumor marker Weihua Hu a,b,c,⇑, Hongming Chen a,b, Zhuanzhuan Shi a,b, Ling Yu a,b a

Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy, Southwest University, Chongqing 400715, China Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, Southwest University, Chongqing 400715, China c Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Wuhan University, Wuhan 430072, China b

a r t i c l e

i n f o

Article history: Received 22 January 2014 Received in revised form 20 February 2014 Accepted 22 February 2014 Available online 4 March 2014 Keywords: Surface plasmon resonance imaging (SPRi) Gold nanoparticle Atom transfer radical polymerization Signal amplification Tumor marker

a b s t r a c t Surface plasmon resonance imaging (SPRi) is an intriguing technique for immunoassay with the inherent advantages of being high throughput, real time, and label free, but its sensitivity needs essential improvement for practical applications. Here, we report a dual signal amplification strategy using functional gold nanoparticles (AuNPs) followed by on-chip atom transfer radical polymerization (ATRP) for sensitive SPRi immunoassay of tumor biomarker in human serum. The AuNPs are grafted with an initiator of ATRP as well as a recognition antibody, where the antibody directs the specific binding of functional AuNPs onto the SPRi sensing surface to form immunocomplexes for first signal amplification and the initiator allows for on-chip ATRP of 2-hydroxyethyl methacrylate (HEMA) from the AuNPs to further enhance the SPRi signal. High sensitivity and broad dynamic range are achieved with this dual signal amplification strategy for detection of a model tumor marker, a-fetoprotein (AFP), in 10% human serum. Ó 2014 Elsevier Inc. All rights reserved.

Surface plasmon resonance imaging (SPRi)1 is a local refractive index-sensitive optical technique showing compelling capability to analyze biomolecular interactions and detect biomolecules with the inherent advantages of being high throughput, label free, and real time [1–5]. During recent years, it has received tremendous research interest as a powerful tool not only for fundamental research but also in some practical fields such as medical diagnostics, food safety, and environmental monitoring [1,5–8]. However, the sensitivity of SPRi, which relies on reflectivity interrogation mode, is even lower than that of an angular-scanning or wavelength-scanning SPR platform [9,10]. The insufficient sensitivity seriously limits various practical applications of SPRi, and intensive research works have been conducted to boost its sensitivity with encouraging progress. Configuration-innovated instruments, such as phase-sensitive SPR/ ⇑ Corresponding author at: Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy, Southwest University, Chongqing 400715, China. E-mail address: [email protected] (W. Hu). 1 Abbreviations used: SPRi, surface plasmon resonance imaging; ATRP, atom transfer radical polymerization; AuNP, gold nanoparticle; DTBE, bis[2-(20 -bromoisobutyryloxy)ethyl]disulfide; AFP, a-fetoprotein; IgG, immunoglobulin G; DMF, N,N-dimethylformamide; BSA, bovine serum albumin; PBS, phosphate-buffered saline; POEGMA-co-GMA, poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate]; Bipy, 2,20 -bipyridyl; CuBr2, copper(II) bromide; HEMA, 2-hydroxyethyl methacrylate; UV–Vis, ultraviolet–visible; DLS, dynamic light scattering; TEM, transmission electron microscope; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CBB, Coomassie Brilliant Blue R-250. http://dx.doi.org/10.1016/j.ab.2014.02.022 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.

SPRi, and specifically designed substrates, such as etched/patterned gold chips and Ag/Au bimetallic chips, have been demonstrated with improved sensitivity [2,10–13]. However, these strategies suffer from sophisticated instrumental setup and/or costly microfabrication processes. Another universal route toward sensitive SPR/SPRi relies on specific reaction/bioconjugation to amplify the weak signal originated from biomolecular interaction for high sensitivity [14]. The core idea is to intensify the local refractive index change by introducing nanomaterials or reaction products onto the sensing surface after the affinity interactions. In this regard, biomolecular conjugated nanomaterials, such as silica nanoparticles (SiNPs), colloidal metal nanostructures, magnetic nanoparticle (MNPs), and quantum dots (QDs), have been employed for signal amplification of SPR/SPRi [3,4,15–18]. Enzyme-catalyzed precipitation has also been developed to amplify the SPRi signal for immunoassay of protein biomarker with RNA aptamer microarray [3]. By building a dynamic equilibrium between the active radicals and dormant species, the living/controlled atom transfer radical polymerization (ATRP) technique allows for precise control over the polymer molecular weights and molecular weight distributions [19]. Recently, it was further demonstrated as a novel signal readout and/or amplification method for reliable immunoassay [20–28]. For example, by conjugating the recognition antibody or antibody-attached macromolecule with the ATRP initiator, readout of the immunoassay signal was directly accomplished by naked

Dual signal amplification for SPRi immunoassay / W. Hu et al. / Anal. Biochem. 453 (2014) 16–21

eyes or thickness measurement after the polymerization [20,21,28]; ATRP was also employed for signal amplification in electrochemiluminescence (ECL) and electrochemical platform [22,23,27]. Recently, an initiator-grafted gold nanoparticles (AuNPs) technique was reported for signal amplification on a singlechannel SPR platform [29,30] However, the initiator and recognition antibody are simultaneously grafted onto the AuNPs, which may result in large variation of the initiator/protein ratio and potential phase separation and/or detachment of the ligands, thus damaging the reliability and reproducibility of the signal amplification. Here, we report a dual SPRi signal amplification by combining functional AuNPs with controllable on-chip ATRP for sensitive SPRi immunoassay. In this strategy, AuNPs were conjugated with bis [2(20 -bromoisobutyryloxy)ethyl]disulfide (DTBE), an ATRP initiator, by Au–S bond to form the AuNPs@DTBE, and the secondary antibody was further absorbed onto the AuNPs@DTBE via ion pair interaction and hydrogen bond (Scheme 1A) to form AuNPs@DTBE–antibody conjugates. After the capture of target on the sensing surface, the AuNPs@DTBE–antibody conjugates were flowed onto the chip surface to form immunocomplexes for first SPRi signal amplification. On-chip ATRP was then triggered by introducing monomer and catalysts for second signal amplification (Scheme 1B). This dual signal amplification strategy was validated on a low-fouling SPRi chip with a-fetoprotein (AFP) as a model biomarker, achieving sensitive immunoassay with excellent specificity, low detection limit, and broad dynamic range in serum sample. Materials and methods Chemicals and materials Mouse anti-AFP monoclonal antibody and AFP were purchased from Abcam. Rabbit anti-AFP polyclonal antibody and anti-rabbit immunoglobulin G (IgG) were purchased from Sangon Biotech. Chloroauric acid (HAuCl4), N,N-dimethylformamide (DMF), and bovine serum albumin (BSA) were purchased from Aladdin. All other chemicals were purchased from Sigma. All chemicals were used without further purification. The buffer used in this work was 0.01 M phosphate-buffered saline (PBS, pH 7.4). All solutions were prepared with deionized

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water from a Millipore Milli-Q system. Protein solutions were stored at 4 °C before use. All experiments were conducted at room temperature. Preparation of AuNPs@DTBE–antibody conjugates Citrate-capped AuNPs were synthesized by the well-established citrate reduction method [31] and stored at 4 °C before use. ATRP initiator grafted AuNPs (AuNPs@DTBE) were prepared by slowly adding 20 ll of DTBE solution (0.1% [v/v] in DMF) into 1 ml of nitrogen-saturated citrate-capped AuNPs solution. The solution was protected from light and gently stirred for 1 h. To prepare AuNPs@DTBE–antibody conjugates, 500 ll of the AuNPs@DTBE solution was slowly added into 1.0 ml of anti-rabbit IgG solution (0.1 mg ml1) in 0.01 M PBS buffer. The mixture solution was incubated at 37 °C for 1 h and then centrifuged for 10 min at 12,000 rpm three times (washed with 0.1% BSA in 0.01 M PBS). Finally, AuNPs@DTBE–antibody conjugates were dispersed in the PBS buffer with 0.1% BSA. Preparation of SPRi chip The SPRi chip was constructed based on the poly[oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate] (POEGMA-coGMA) brush, which was described in our previous publication [1,32]. A clean SPRi chip with a 4  4 patterned spot array was incubated with a 1.0-mg ml–1 cysteamine ethanol solution for 24 h, followed by intensive ethanol rinsing. After drying, the chip was dipped in a 10-ml tetrahydrofuran (THF) solution containing 64 ll of 2-bromoisobutyryl bromide (BIB) and 77 ll of triethylamine (TEA) for 2 h of incubation in an ice bath, followed by rinsing with ethanol. The chip with initiators was further dipped into a 25ml ATRP growth solution (1:1 H2O/methanol solution with 2.3 mg ml1 2,20 -bipyridyl (Bipy), 1.68 mg ml1 copper(II) bromide (CuBr2) 10% (v/v) oligo(ethylene glycol) methacrylate (OEGMA, Mn = 360), and 0.5% (v/v) glycidyl methacrylate (GMA) in a 50ml centrifuge tube. Then, 65 mg of l-ascorbic acid in 1 ml of water was rapidly added into the solution to trigger the surface-initiated ATRP. The tube was sealed and kept in a nitrogen atmosphere for 6 h. After the growth of polymer brush, the chip was transferred to a 0.1-M sodium azide solution to quench the remaining

Scheme 1. (A) Preparation of AuNPs@DTBE–antibody. (B) Dual SPRi signal amplification. Sample was flowed on the SPRi chip surface to directly detect AFP target (step I), followed by AuNPs@DTBE–antibody to form immunocomplexes for the first signal amplification (step II), and on-chip ATRP was triggered to further enhance the signal (step III).

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initiators on the surface [33] and was washed with ethanol and H2O and dried with a gentle nitrogen flow. To immobilize antibody onto chip, approximately 0.2 ll of antibody solution (200 lg ml–1 mouse anti-AFP in 0.01 M PBS) was manually dropped with a pipette onto the prepatterned gold spot of the POEGMA-co-GMA modified chip. The chip with antibody solution was dried in a dry cabinet for 12 h, followed by thorough rinsing with 0.01 M PBS and drying with a gentle nitrogen flow for detection. SPRi detection and signal amplification All SPRi measurements were performed on a GWC SPRimager II system. First, 10% human serum was prepared by diluting human serum (from clotted human male whole blood; Sigma) with 0.01 M PBS. The sample solution was prepared by spiking AFP target into 10% human serum. The sample solution was first flowed to the chip surface at 150 ll min1 for 1 h to capture the AFP (step I in Scheme 1B), followed by a polyclonal rabbit anti-AFP solution (10 lg ml–1) for 15 min at the same flow rate. After washing with 0.01 M PBS, AuNPs@DTBE–antibody solution was injected at 150 ll min1 and incubated for 1 h and then washed with 0.01 M PBS (step II in Scheme 1B). For second signal amplification, ATRP growth solution containing 13.5 ml of water, 1.5 ml of methanol, 10.0 mg of CuBr2, 14 mg of Bipy, 1.5 ml of 2-hydroxyethyl methacrylate (HEMA), and 30 mg of just-added L-ascorbic acid was flowed to the sensing surface at 150 ll min1 for 5 min (step III in Scheme 1B). Characterizations Ultraviolet–visible (UV–Vis) spectra were collected by a UV2550 spectrometer, and Fourier transform infrared (FTIR) spectra were collected by a Nicolet FTIR 8700 instrument. Dynamic light scattering (DLS) data were obtained by a Malvern Zeta Analyzer ZEN3600 instrument. Transmission electron microscope (TEM) images were collected on a JEM-1400 Transmission Electron Microscopy system (JEOL, Japan) operated at 100 kV. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was performed on a Mini-PROTEAN Tetra System. AuNPs@DTBE –anti-rabbit IgG conjugate, AuNPs@DTBE, and citrate-protected AuNPs were heated at 95 °C for 5 min in 5% (v/v) 2-mercaptoethanol-containing sample buffer before analysis on 8% gels by SDS–PAGE. The gels were stained in Coomassie Brilliant Blue R250 (CBB) stain (0.25% in 46% ethanol containing 9.2% acetic acid) for 2 h and then destained in 25% ethanol with 10% acetic acid until a clear background was reached. Results and discussion Via ligand exchange, citrate-capped AuNPs were first grafted with the ATRP initiator DTBE, which possesses high affinity to the gold surface due to its disulfide group. Anti-rabbit IgG was further grafted onto the AuNPs@DTBE to form AuNPs@DTBE–antibody conjugate. The assembly is possibly driven by the hydrogen bond between the carbonyl group of DTBE and the amide group of antibody and/or ion pair interaction between the bromine atom of DTBE and the antibody [34]. Because AuNPs demonstrate characteristic visible light adsorption around 520 nm due to their inherent local SPR, UV–Vis spectra of AuNPs before and after modification were recorded to monitor the graft process, as shown in Fig. 1. The pristine AuNPs stabilized by citrate exhibit a strong adsorption around 519.5 nm, indicating an average diameter of 16 nm, which is consistent with the TEM observation shown in the inset of Fig. 1. After the graft of DTBE, the adsorption peak shifts

Fig.1. UV–Vis spectra of citrate-protected AuNPs (a), AuNPs@DTBE (b), and AuNPs@DTBE–antibody conjugate (c). The inset shows the TEM image of citrateprotected AuNPs.

to 521.5 nm, originated from the change of local dielectric constant of AuNPs on DTBE adsorption. A 5-nm red shift to 526.5 nm is further observed when AuNPs@DTBE is incubated with anti-rabbit IgG antibody solution, which is in line with previous report, suggesting the successful graft of the antibody on the AuNPs@DTBE [35]. Size distributions of the AuNPs, AuNPs@DTBE, and AuNPs@DTBE–antibody were collected by DLS measurement, as shown in Fig. 2. Citrate-capped AuNPs demonstrate a diameter distribution centered at 25 nm, which is slightly larger than that obtained from UV–Vis and TEM measurements, probably due to the hydrated layer on the AuNPs [35]. After graft of DTBE initiator and subsequent antibody, the average diameter increases to 38 and 59 nm, respectively, further confirming the successful preparation of AuNPs@DTBE–antibody conjugate. The AuNPs@DTBE–antibody conjugate was analyzed by SDS– PAGE. In CBB-stained gel as shown in Fig. 3, there are two bands with molecular weights of approximately 53 and 25 kDa for antirabbit IgG in lane e, corresponding to the heavy chain and light chain of the antibody [36]. The same two bands are observed for AuNPs@DTBE–anti-rabbit IgG conjugate (lane b), but not for either AuNPs@DTBE (lane c) or citrate-capped AuNPs (lane d), unambiguously unveiling that the anti-rabbit IgG was grafted onto AuNPs@DTBE. SPRi detection was performed in 10% human serum with AFP as a model target. AFP is an important tumor marker closely associated with diagnosis of hepatocellular carcinoma and other chronic liver diseases. The commonly used cutoff concentration of AFP for early screening and diagnosis of tumor is 20 ng ml1 in serum [37,38]. We first tried direct detection of 10 ng ml1 AFP in serum, and the SPRi difference image and its line profile are shown in Fig. 4A. It is observed that the SPRi signals on all of the sensing spots (with preattached mouse anti-AFP, indicated with a red rectangle) and control spots remain negligible, very close to the noise level of SPRi measurement, indicating that with 10 ng ml1 AFP it is very hard to evoke a detectable signal on the SPRi platform by using direct detection. It is also strongly indicative of the low-fouling nature of the SPRi chip built on the POEGMA-co-GMA brush if considering that the sample is 10% human serum, which contains abundant nonspecific proteins. When the AuNPs@DTBE–anti-rabbit IgG conjugate was flowed on the SPRi chip after a 10-min rabbit anti-AFP incubation, the SPRi

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Fig.2. DLS-determined hydrodynamic diameter distribution plots of citric-protected AuNPs (A), AuNPs@DTBE (B), and AuNPs@DTBE–antibody (C).

Fig.3. SDS–PAGE analysis of AuNP and its antibody conjugate. Lane a: molecular weight marker; lane b: AuNPs@DTBE–anti-rabbit IgG conjugate; lane c: AuNPs@DTBE; lane d: citrate-protected AuNPs; lane e: pure anti-rabbit IgG.

signals on the AFP sensing spots are significantly enhanced to an average SPRi intensity of 4.09 ± 0.41 pixels, as shown in Fig. 4B. However, the signals on the control spots remain as low as 1.58 ± 0.26 pixels, which may be originated from the change of bulk solution. Thus, the sensing spots are easily differentiated from

the control spots, suggesting that 10 ng ml1 AFP is detectable with the signal amplification of AuNPs@DTBE–anti-rabbit IgG conjugate. Fig. 5 shows the representative in situ SPRi response for AuNPs@DTBE–anti-rabbit IgG conjugate on sensing spots that are prereacted with samples containing AFP of different concentrations. It is exhibited that the SPRi signal increases in an AFP concentrationdependent manner and that higher AFP concentrations cause higher increases of SPRi signal. During this process, the high affinity between the conjugated anti-rabbit IgG and underlying rabbit anti-AFP directs the AuNPs@DTBE–anti-rabbit IgG to bind onto the sensing surface, resulting in effective signal enhancement due to the high mass density and dielectric constant of AuNPs and electromagnetic coupling between AuNPs and the surface plasmon wave on the SPRi chip [39–41]. Notably, on the negative control spot without captured AFP, the SPRi signal remains stable and low, consistent with the result in Fig. 4B, confirming that the first signal amplification is specific for target AFP. On-chip ATRP was employed to further enhance the SPRi signal. When the ATRP growth solution containing monomer (HEMA) and catalyst (Cu(I)Bipy2) was flowed to the chip, ATRP of HEMA was triggered and PHEMA polymer chains with high density were controllably grown from the initiator-attached AuNPs, generating secondary SPRi signal amplification. As shown as the SPRi different image and the line profile in Fig. 4C, the SPRi signal on sensing spots for 10 ng ml1 AFP is enhanced to 9.95 ± 0.76 pixels, whereas the signal for negative control is only 2.21 ± 0.37 pixels, indicating that the on-chip polymerization is effective for signal amplification with a satisfying specificity. The dose responses for AFP detection in 10% serum using this signal amplification strategy are plotted in Fig. 6, where each data point with a standard deviation error bar is obtained by averaging

Fig.4. SPRi images and line profiles for detection of 10 ng ml1 AFP in 10% human serum: (A) direct detection; (B) after signal amplification with AuNPs@DTBE–antibody conjugate; (C) after dual signal amplification with AuNPs@DTBE–antibody conjugate and subsequent on-chip ATRP. The four spots in the red rectangle on the chip are spotted with anti-AFP, whereas other spots are spotted with BSA as control. (For interpretation of the reference to color in this figure legend, the reader is referred to the Web version of this article.).

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Fig.5. In situ SPRi responses for AuNPs@DTBE–anti-rabbit IgG conjugate. The chip is preincubated in AFP-contained sample, and the AFP concentration is indicated in the figure.

the responses from 12 spots in three independent chip measurements. With AuNPs@DTBE–antibody conjugate alone for signal amplification, the detection limit (defined as the concentration that produces signal higher than Mb + 3Sd, where Mb is the mean signal for negative control and Sd is the standard deviation) [42], reaches 5.0 ng ml1 (Fig. 6A) and the detection limit is further down to 1.0 ng ml1 when the dual signal amplification based on AuNPs@DTBE–antibody conjugate and subsequent on-chip polymerization is applied. It is found in Fig. 6B that the data points for high concentrations (i.e., 5.0, 10, and 20 ng ml1) deviate from the linear relation, which may be originated from the lower rate of on-chip polymerization due to higher density of AuNPs on the chip surface. For practical application, these concentrations could be directly quantified with one-step signal amplification as in Fig. 6A. As mentioned above, the cutoff values of most biomarkers for early tumor screening are in the several to tens of ng ml1 range [43]; therefore, the reported strategy allows SPRi immunoassay chip to fully meet the clinical requirements. Depending on the target concentration in the sample, direct detection one-step amplification and dual signal amplification could be used, thereby enabling quantification of the target marker in a wide concentration range. The reported strategy is also applicable to other SPR platforms such as angular-scanning and wavelength-interrogated SPR because they share the same principle with SPRi. Conclusion In this work, a dual signal amplification strategy has been developed for an SPRi chip to sensitively and specifically detect tumor marker in human serum. AuNPs conjugated with ATRP initiator DTBE and secondary antibody (AuNPs@DTBE–antibody) are used to generate the first signal amplification, and subsequent on-chip polymerization of HEMA is employed to further intensify the SPRi signal. With this strategy, sensitive immunoassay of AFP as a model biomarker is demonstrated, showing a detection limit as low as 1.0 ng ml1 and a broad dynamic range covering 1.0 ng ml1 to higher concentrations. The reported strategy offers a potentially powerful solution for sensitive SPRi immunoassay in a wide diversity of practical fields. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21205098 and 21273173), the Natural Science Foundation Project of CQ CSTC (cstc2012jjA10099), the Key Laboratory of Analytical Chemistry for Biology and Medicine (Wuhan University), the Ministry of Education (ACBM2012006), the Institute for Clean Energy and Advanced Materials (Southwest University), the Chongqing Key Laboratory for Advanced Materials and Technologies of Clean Energies, a start-up grant under SWU111071 from Southwest University, the Chongqing Engineering Research Center for Rapid Diagnosis of Dread Disease, and the Chongqing Development and Reform Commission. References

Fig.6. Dose–response plots for AFP detection in 10% serum curves: (A) with signal amplification by AuNPs@DTBE–antibody alone; (B) with dual signal amplification by AuNPs@DTBE–antibody and subsequent ATRP.

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