Ultrasensitive, Multiplex Raman Frequency Shift ... - ACS Publications

Jan 5, 2016 - Shift Immunoassay of Liver Cancer Biomarkers ... Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China...
0 downloads 0 Views 7MB Size
Download PDF
Ultrasensitive, Multiplex Raman Frequency Shift Immunoassay of Liver Cancer Biomarkers in Physiological Media Bochong Tang,† Jiaojiao Wang,†,‡ James A. Hutchison,§,∥ Lei Ma,† Ning Zhang,⊥ Hua Guo,⊥ Zhongbo Hu,‡ Min Li,*,† and Yuliang Zhao*,† †

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, 19B, Yuquan Road, Shijingshan District, Beijing 100049, China ‡ College of Materials Science and Optoelectronic Technology, University of Chinese Academy of Sciences, 19A, Yuquan Road, Shijingshan District, Beijing 100049, China § ISIS & icFRC, University of Strasbourg and CNRS, 8 allée Gaspard Monge, 67000 Strasbourg, France ∥ School of Chemistry and Bio21 Institute, University of Melbourne, Victoria 3010, Australia ⊥ Department of Cancer Cell Biology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China S Supporting Information *

ABSTRACT: Highly sensitive multiplex biomarker detection is critical for the early diagnosis of liver cancer. Here, a surface-enhanced Raman scattering (SERS) frequency-shift immunoassay is developed for detection of liver cancer biomarkers α-fetoprotein and Glypican-3 down to subpicomolar concentrations in saline solution. A high temperature modification of the Tollen’s method affords silver nanoparticle films with excellent SERS response upon which ordered domains of Raman reporters are chemisorbed by microcontact printing. Shifts in the reporters SERS spectrum in response to a bound antibody’s biomarker recognition constitutes the frequency shift assay, exhibiting here exceptional sensitivity and specificity and shown to function in fetal calf serum and in the serum of a patient with hepatocellular carcinoma. KEYWORDS: frequency shift immunoassay, microcontact printing (μCP), multiplex detection, SERS, liver cancer biomarkers prevention,5 the development of sensitive and specific methods for early diagnosis remains a matter of urgency.2,6−8 α-Fetoprotein (AFP) is an established biomarker for early diagnosis of the most common form of liver cancer, hepatocellular carcinoma (HCC).2,9−15 Unfortunately, even for a low threshold of 11 ng/mL (150 pM) AFP in serum, one-third of HCC sufferers go undiagnosed, while higher levels of AFP can

L

iver cancer accounted for the second most deaths due to cancer worldwide in 2012, its high mortality-to-incidence ratio (>0.95) due to the late manifestation of clinical symptoms, and to incomplete understanding of its pathogenesis.1,2 It is one of only a few cancers for which there has been no improvement in prognosis in recent decades.3 Globally, the majority of cases occur in Africa and Asia, 50% of cases in China alone, reflecting in part the prevalence of viral hepatitis (B or C), which is a major risk factor.1,2,4 Although vaccination against, and management of, viral hepatitis is at the frontline of liver cancer © XXXX American Chemical Society

Received: September 23, 2015 Accepted: January 5, 2016

A

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

www.acsnano.org

Article

ACS Nano

Figure 1. (a) Photograph of a silver nanoparticle SERS substrate (ca. 1 cm2) used herein, following suspension in glutaraldehyde/ammoniacal silver solutions for 4 min at 95 °C (schematic inset). (b) The extinction spectrum (−log10(T)) calculated from the transmittance (T) of the silver nanoparticle film on glass. (c),(d) SEM images of the silver nanoparticle film in (a) (the SERS substrates used herein). (e) SEM image of a silver nanoparticle film following suspension in glutaraldehyde/ammoniacal silver solutions for 2 h at room temperature. (f) SEM image of a PDMS stamp for microcontact printing of Raman reporter MBA in 2 × 2 μm squares on the silver nanoparticle substrates. Inset: Lateral Force Microscopy image showing the integrity of domains of 11-aminoundecanethiol chemisorbed to atomically flat gold using the PDMS stamp.

multiplex assays were recently developed using ELISA and chemiluminescence.11,15 Immunoassays based on SERS have potential for increased sensitivity over conventional ELISA and for low-interference multiplex sensing due to their highly resolved spectra compared to luminescence assays.8 SERS spectroscopy utilizes enhanced optical excitation and scattering at metal surfaces associated with surface plasmon polaritons (SPPs), the coherent coupling of light with the free electron plasma of the metal. In a ca.1 nm gap between two 25 nm silver nanoparticles, Raman scattering can be enhanced by as much as 1011 making single molecule SERS

occur in cirrhosis and hepatitis sufferers, making ultrasound monitoring of at-risk patients the more reliable approach.9,13−15 In order to improve HCC detection sensitivity and specificity, a range of other biomarkers have been sought.9−13,15 Glypican-3 (GPC3), a heparan sulfate proteoglycan involved in cellular growth, is one promising example.2,11,15 Serum GPC3 has good sensitivity (40−60%) and excellent specificity (>90%) for HCC and when detected in combination with AFP can increase the sensitivity of HCC diagnosis to 80%.11 Although there are doubts about the utility of GPC3 for HCC diagnosis,12 AFP/GPC3 B

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Scheme 1. Sequential Chemisorption of the MBA and DSNB-Derived Thiolate Reporters in Domains on the Silver Nanoparticle Substrate, Followed by Covalent Binding of Anti-AFP and Anti-GPC3 Antibodies, and the Subsequent Capture of the Corresponding Antigens AFP and GPC3

2 orders of magnitude (10−13 M). Furthermore, we employ microcontact printing to define ordered domains of chemisorbed Raman reporters on the substrate, greatly reducing interference for sensing of one biomarker in the presence of the other. Finally we demonstrate the assay in both a model physiological medium (fetal calf serum) and in the serum of an HCC sufferer, showing the potential utility of this sensing modality in clinical settings.

sensing feasible.16 However, SERS sensing of biomolecules remains challenging due to their often large size relative to SERS “hotspot” dimensions and their potential lack of highly polarizable (Raman active) moieties. Indirect detection of biomolecules by labeling with a Ramanactive reporter molecule is an effective alternative; for instance, sandwich structures consisting of metal-biomolecule-Raman reporter-metal create SERS hotspots coinciding with a biomolecule detection event.17−26 These methods can suffer however from nonspecific adsorption of the Raman label due to multiple immunoreaction steps.27−29 A novel indirect approach introduced by Olivo and coworkers, SERS “nanostress” sensing, monitors frequency shifts in a Raman reporter vibrational modes in response to changes in its chemical environment or orientation (here the binding of a target biomolecule)30,31 with the technique also being employed recently for small molecule sensing.32,33 The single immunoreaction step required decreases the influence of nonspecific protein adsorption. Olivo used the frequency shift immunoassay to detect two different proteins separately using a mixed monolayer of Raman reporters on a metallic substrate;30 however, significant cross-talk (up to 34%) was observed, a serious obstacle for multiplex detection. The best sensitivity exhibited to date for frequency-shift sensing of a protein is 10 pM.34 Here, we extend multiplex SERS frequency-shift immunoassay sensitivity and specificity to unprecedented levels. We develop a wet chemical silver nanoparticle film fabrication for SERS substrates with excellent response, improving assay sensitivity by

RESULTS AND DISCUSSION Silver nanoparticle films for SERS substrates were grown on mercaptosilanized slides (silicon or glass) using a high temperature modification of the Tollen’s method (Experimental section). In the classical method, substrates are developed in an ammoniacal silver solution in the presence of a reductant (here, glutaraldehyde) at temperatures from ambient up to 60 °C and times up to days, resulting in deposition of typically globular silver islands (see for example Figure 1e). Here, the reaction was instead rapidly heated to 95 °C with the substrate suspended within for 4 min without stirring, resulting in a dense coating of highly faceted ∼45 nm diameter silver particles uniform on the cm scale and stable against tape peeling (photograph Figure 1a and SEM images Figure 1c,d). An extinction spectrum of the silver nanoparticle film is shown in Figure 1b, taken in transmission on glass. The minimum at ca. 326 nm is the bulk plasma frequency of silver, the shoulder at ca. 420 nm is attributed to localized SPPs of the individual silver nanoparticles, and the broad extinction across the visible peaking at ca. 770 nm to coupling of localized SPPs, so-called gap modes associated with the highest intensity SERS hot-spots.16 The silver film C

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 2. (a) Raman spectra of a DSNB/anti-AFP bound substrate after exposure to phosphate-buffered saline solutions of AFP of various concentrations (offset for clarity). (b) Zoom-in of the spectral region around 1335 cm−1. (c) Semilog plot of the absolute peak shift as a function AFP concentration for the ca. 1335 cm−1 peak. (d) Raman spectra of a MBA/anti-GPC3 bound substrate after exposure to PBS solutions of GPC3 of various concentrations (offset for clarity). (e) Zoom-in of the spectral region around 1585 cm−1. (f) Semilog plot of the absolute peak shift as a function of GPC3 concentration for the ca. 1585 cm−1 peak. Error bars in (c) and (f) indicate the standard deviation of five measurements and the solid lines are linear fits.

should thus be an excellent SERS substrate using 780 nm laser

(a DSNB-derived thiolate, vide infra) recorded at different places on one substrate was 6% and from sample to sample was 10%. The average SERS enhancement factor (following Le Ru and Etchegoin16) for the substrates was estimated to be 107

excitation. The relative standard deviation (averaged across several peaks) of SERS intensity from a monolayer of a Raman reporter D

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

respectively averaged over five measurements. We estimate an uncertainty for the measured frequency shifts of ±0.3 cm−1. Following Olivo, the shifts are attributed to a change in the chemical environment of the DSNB-derived reporter upon antigen binding, resulting in normal mode SERS frequency shifts. Specifically, Olivo and co-workers attributed a downshift in the Raman peak occurring upon antigen binding to the mechanical relaxation of tensile stress in the Raman reporter molecule which is first imposed by steric repulsions when the reporter monolayer binds the antibody.30 The relaxation of steric repulsions was attributed to attractive hydrophobic interactions between bound antigens allowing more efficient packing. The shifts in the Raman peak observed here show a similar trend, for instance the ca. 1335 cm−1 peak of the DSNB-derived reporter monolayer was upshifted by 0.7 cm−1 upon conjugation with the anti-AFP, before downshifting again when AFP was present in progressively higher concentrations. Alvarez-Puebla and coworkers emphasize that changes in the orientation of the reporter relative to the substrate, with corresponding changes in Raman selection rules, could also effectively sense antigen binding.31 Sensing of GPC3 over the range 10−13 M to 10−10 M in PBS was also performed on an unpatterned MBA/anti-GPC bound substrate (Figure 2d,e). The SERS spectrum of MBA has an intense peak near 1585 cm−1 corresponding to the symmetric aromatic C−C stretch. The peak shifted to lower wavenumbers by 2.5 ± 0.3 cm−1 and 0.6 ± 0.1 cm−1 when the substrate was exposed to 10−10 M and 10−13 M GPC3 solutions, respectively, showing again a linear semilog relationship over this range (Figure 2f). Simultaneous AFP and GPC3 detection from PBS solutions was carried out using the patterned substrate described earlier, the 10 μm diameter laser spot allowing Raman signals from both DSNB/anti-AFP and MBA/anti-GPC3 bound domains to be detected simultaneously (see Figure 1f, inset). Figure 3a shows the SERS spectrum from the substrate after immersion in equimolar mixtures of AFP and GPC3 in PBS with the concentration of each protein ranging from 10−10 to 10−13 M, and a blank. The DSNB-derived thiolate peak at ca. 1335 cm−1 and the MBA peak at ca. 1585 cm−1 are shown in Figure 3b and c, respectively. Peak shifts of similar direction and magnitude occur for this multiplex experiment as were observed in the experiments on the individual reporters, confirming the integrity of the patterned domains of the respective antibody/reporter conjugates (for AFP detection, the ca. 1335 cm−1 peak downshifted by 3.2 ± 0.3 cm−1 for the 10−10 M equimolar protein solution and by 0.7 ± 0.3 cm−1 for the 10−13 M solution; for GPC3 detection, the ca. 1585 cm−1 peak downshifted by 2.8 ± 0.3 cm−1 for the 10−10 M solution and by 0.7 ± 0.3 cm−1 for the 10−13 M solution). To ensure that the Raman peak shifts of the DSNB-derived thiolate and MBA reporters were specifically due to AFP and GPC3 binding events respectively, the patterned substrate with DSNB/anti-AFP and MBA/anti-GPC3 bound regions were exposed to solutions of only one biomarker solution, AFP or GPC3, and SERS spectra recorded (Figure 4). Clearly, when only AFP is present in solution the DSNB-derive thiolate peak shifts as anticipated, whereas the MBA peak is not shifted beyond ±0.3 cm−1, and vice versa. Thus, there is no discernible cross-talk between the two biomarker detection channels beyond the uncertainty. The true test of any immunoassay is its performance in physiologically relevant media. To assess this, we first repeated the simultaneous sensing of AFP and GPC3 using the patterned

compared to the same reporter monolayer on atomically flat gold. The multiplex biomarker detection regime is outlined in Scheme 1. Poly(dimethylsiloxane) (PDMS) stamps were used to chemisorb the Raman reporter mercaptobenzoic acid (MBA) in 2 μm × 2 μm square domains on the Ag substrate using microcontact printing (μCP) (Experimental Methods and SEM image, Figure 1f). The substrate was rinsed to remove unbound MBA then soaked overnight in an acetonitrile solution of a second reporter 5,5′-dithiobis(succinimidyl-2-nitrobenzoate) (DSNB) which bound to the regions between the MBA domains. The integrity of domain transfer was checked by chemisorbing 11-aminoundecanethiol (AUT) to an atomically flat gold surface, followed by lateral force microscopy imaging (inset, Figure 1f). In all cases, chemisorption occurs via Ag−S bonding, and follows cleavage of the disulfide bond in the case of DSNB.35 The substrate bound with domains of MBA and DSNBderived thiolate was immersed in a phosphate-buffered saline (PBS) solution containing AFP monoclonal antibody (antiAFP). Binding of anti-AFP occurs via primary amine nucleophilic attack on the succinimidyl ester of the DSNB-derived thiolate. After blocking unreacted DSNB-derived thiolate by the same reaction with bovine serum albumin (BSA), the substrate was immersed in a solution of ethyl(dimethylaminopropyl) carbodiimide/N-hydroxysuccinamide (EDC/NHS) to activate the carboxyl groups on MBA for binding with GPC3 monoclonal antibody (anti-GPC3). After soaking in anti-GPC/PBS solution the substrate was again blocked by BSA and thoroughly rinsed. For multiplex sensing, the patterned antibody-conjugated Ag substrates were immersed in PBS solutions, or sera, containing mixtures of AFP and GPC3, resulting in antibody/antigen recognition, and SERS spectra collected from a 10 μm diameter focal spot which overlapped both domains (Figure 1f and see Experimental Methods). In experiments where one antigen alone was measured, unpatterned substrates could be employed. In that case the same frequency shifts were observed as for patterned substrates during single antigen sensing except the Raman intensity was higher due to the laser focus being filled by the single relevant reporter/antibody. In initial experiments AFP and GPC3 were sensed separately from PBS solutions using unpatterned silver substrates. Figure 2a,b shows the SERS spectra (offset for clarity) of a DSNB/antiAFP bound substrate following immersion in AFP/PBS solutions with concentrations ranging from 10−10 M to 10−13 M, together with PBS without AFP (blank). The SERS spectrum of DSNB-derived thiolate is dominated by an intense band near 1335 cm−1 corresponding to the symmetric nitro stretch.35 This peak clearly shifts to lower wavenumber as a function of exposure to AFP solutions of different concentration, and thus with increasing equilibrium concentration of bound antigen, exhibiting an exponential relationship over the range studied (Figure 2b,c). At AFP concentrations >10−9 M no further shift could be discerned indicating saturation (see Supporting Information (SI) Figure S1). The spectra were recorded at 1 cm−1 intervals and the peak position extracted either by fit to a b-spline function, or to voigt profiles after background subtraction, giving similar results. The peak position of the reporter on one substrate in the absence of AFP varied by less than 0.1 cm−1 over repeated measurements, whereas the absolute peak shift was 2.4 cm−1 ± 0.3 cm−1 and 0.7 ± 0.2 cm−1 for a 10−10 M and a 10−13 M AFP solution, E

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

10−10 M, with the same trends of downshifts in the respective DSNB and MBA peaks observed as for PBS, however, with more uncertainty at the 10−13 M level (SI, Figure S2). For instance for the equimolar 10−13 M AFP/GPC3 solution in this experiment, exactly the same downshift of the MBA peak was observed in fetal calf serum as for PBS, however the DSNB-derived thiolate peak did not shift. The increased uncertainty is attributed to nonspecific interactions between anti-AFP and fetal calf serum proteins. The ultimate relevance for this immunoassay however is its sensing capability in human sera. Through the Tianjin Medical University Cancer Institute and Hospital, China, we obtained a 50 μL serum sample from a patient with hepatocellular carcinoma upon which we undertook multiplex sensing using the patterned DSNB/anti-AFP and MBA/anti-GPC3 bound substrate as above. Typical for a patient with symptomatic HCC, the serum had elevated levels of AFP (8507 ng/mL, 1.2 × 10−7 M) and GPC3 (18.3 ng/mL, 5.0 × 10−10 M) as determined by ELISA. We diluted the sample 100× with fetal calf serum to achieve biomarker levels more relevant to early detection and within the response range of our sensing modality. The results of the frequency shift immunoassay are shown in Figure 5. The downshift in the DNSB-derived thiolate peak was 3.4 cm−1 ± 0.1 cm−1 and for the MBA peak, 1.7 cm−1 ± 0.1 cm−1. Using the Raman peak shift vs biomarker concentration data determined in PBS in Figure 2c and f as standard curves, these shift values correspond to AFP and GPC3 concentrations of 4.3 × 10−9 M and 5.9 × 10−12 M, respectively. Taking into account the 100× dilution, these results correspond accurately with the values measured by ELISA (1.2 × 10−7 M AFP and 5.0 × 10−10 M GPC3). These experiments are thus a proof-of-principle that multiplex SERS frequency shift sensing has potential application for trace biomarker detection in clinical settings.

CONCLUSION In summary, we developed a multiplex SERS frequency shift immunoassay for liver cancer biomarkers exhibiting subpicomolar sensitivity and high specificity for each biomarker in phosphate buffered saline solution. The same multiplex immunoassay undertaken in fetal calf serum showed some interference at the level of 10−13 M for AFP sensing, likely due to nonspecific protein adsorption. Nevertheless, the sensitivity exhibited here is at least 2 orders of magnitude higher than conventional ELISA and more than adequate for discriminating, for instance, baseline serum levels of GPC3 in non-HCC patients (4 ng/mL, ∼ 100 pM) and a typical HCC-positive threshold of 30 ng/mL (∼1 nM).10 Finally, as a proof-of-principle demonstration, we successfully assayed a serum sample from a HCC patient with known, elevated levels of AFP and GPC3 but which was diluted such that the biomarkers were present at concentrations as low as 5 pM. Although such levels are too low to be relevant for early diagnosis of HCC by AFP or GPC3, this work opens possibilities for the simultaneous detection of elevated levels of other biomarkers with subpicomolar normal serum levels (associated with HCC or other diseases). The multiplexing potential of using frequency shifts of highly resolved Raman peaks is key here and microcontact printing is well developed for, for example, chemisorption of more complex patterns of domains of multiple Raman reporters.36−38 For diseases like HCC, where there is significant variability between patients of HCC up-regulated biomarkers, assaying the largest range of relevant biomarkers is

Figure 3. (a) Raman spectra of the composite DSNB/anti-AFP and MBA/anti-GPC3 bound substrate, after exposure to equimolar phosphate buffered-saline solutions of AFP and GPC3 of various concentrations (offset for clarity). (b) and (c) Zoom-ins of the DSNB-derived thiolate peak at ca. 1335 cm−1 (for AFP recognition) and the MBA peak at ca. 1585 cm−1 (for GPC3 recognition), respectively.

substrate but using solutions of fetal calf serum instead of phosphate buffered saline. Equimolar fetal calf serum solutions of AFP and GPC3 were investigated over the range 10−13 M to F

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 4. (a), (d) Raman spectra of the composite DSNB/anti-AFP and MBA/anti-GPC3 bound substrate after exposure to separate phosphatebuffered saline solutions of AFP and GPC3, respectively. (b), (f) Zoom-ins of the DSNB-derived thiolate peak region at ca. 1335 cm−1 in response to separate PBS solutions of AFP and GPC3 solutions, respectively. (c), (e) Zoom-ins of the MBA peak region at ca. 1585 cm−1 in response to separate PBS solutions of AFP and GPC3 solutions, respectively.

(Beyotime Biotechnology), PDMS (Sylgard 184, Dow Corning), and AUT (Dojindo Molecular Technologies, Inc.) were used as obtained. DSNB was synthesized using a reported method.35 Methods. Silver Nanoparticle Films. Silicon (100) wafers or glass slides (1 cm2) were cleaned with piranha solution at 90 °C ∼ 95 °C for 1 h (H2SO4:H2O2 = 3:1, solution should be handled with great care). Substrates were immersed in 3-mercaptopropyl-

critical for reliable early detection and potential improved prognoses.

EXPERIMENTAL METHODS Materials. MBA (Sigma-Aldrich), EDC and NHS (TCI Tokyo), AFP (Genway Biotech), anti-AFP (eBioscience), GPC3 and anti-GPC (Abnova), fetal calf serum (GIBCO), BSA G

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 5. (a) Raman spectra of the composite DSNB/anti-AFP and MBA/anti-GPC3 bound substrate, after exposure to HCC patient serum diluted 100× by fetal calf serum (the blank is the spectrum obtained in pure fetal calf serum). (b) and (c) Zoom-ins of the DSNB-derived thiolate peak region around 1335 cm−1 (for AFP recognition) and of the MBA peak region around 1585 cm−1 (for GPC3 recognition), respectively.

HCC Patient Serum. The serum was provided, with consent of the patient, by Tianjin Medical University Cancer Institute and Hospital.

triethoxysilane (2% in toluene, 12 h) then rinsed (ethanol) and dried (N2(g)). To a 50 mL aqueous 440 mg silver nitrate solution was added dropwise 200 μL 10% aq. NaOH giving a dark green suspension. A maximum 2 mL 10% ammonia was added dropwise over 20 min, until the solution became transparent. The resulting Tollen’s reagent (ammoniacal silver) solution was cooled in an ice−water bath, and the substrate suspended within. Ten drops of aqueous 25% glutaraldehyde were added in 30 s and the solution stirred for 2 min, at which point it turned yellow indicating silver nanoparticle growth. For films with optimal SERS response the solution was rapidly heated to 95 °C with the substrate kept in the reactor for 4 min without stirring before removal and rinsing and storage in ethanediol. Substrate Preparation. PDMS stamps (1 cm2) were prepared by casting Sylgard 184 on a clean silicon template with an embossed 2 μm × 2 μm square pattern (500 nm in depth, 2 μm separation between domains, 1 cm2 array size) followed by curing at 60 °C for 2h. The stamp was soaked in 50 μL of AUT solution (5 mM in ethanol) for 1 min, dried (N2(g)), and contacted to atomically flat gold. After 30 s contact, the stamp was peeled off and the gold rinsed (ethanol) and dried (N2(g)) for lateral force microscopy characterization. The silver nanoparticle films were patterned with arrays of MBA domains in the same way, immersed in 1 mM DSNB/acetonitrile solution overnight, then rinsed (acetonitrile, ethanol, and distilled water). Antibody Binding. The MBA and DSNB-bound substrates were immersed in 0.01 mg/mL anti-AFP in PBS (10 mM, pH 7.4) at 4 °C for 8 h, then immersed in 1 mg/mL BSA solution (PBS, 10 mM, pH 7.4) for 30 min at room temperature, followed by rinsing with 10 mM PBS containing 5% Tween-80 solution (pH 7.4) and 10 mM PBS (pH 7.4). The substrates were then immersed in EDC/NHS (50 mM/10 mM) solution (PBS, 10 mM, pH 6.8) for 1 h at room temperature before soaking in 0.0095 mg/mL anti-GPC3 solution (PBS, 10 mM, pH 7.4) at 4 °C for 2 h. The substrates were then immersed in 1 mg/mL BSA solution (PBS, 10 mM, pH 7.4) for 30 min at room temperature and rinsed with 10 mM PBS containing 5% Tween-80 solution (pH 7.4) and 10 mM PBS (pH 7.4). Sensing. The antibody-bound substrates were immersed in PBS solutions or serum containing AFP and GPC3 for 4 h at room temperature then rinsed with 10 mM PBS containing 5% Tween-80 solution (pH 7.4) and distilled water. SERS spectra were recorded on a DXR Smart Raman spectrometer (Thermo Fisher, 780 nm, 80 mW, 10 μm diameter focal spot laser excitation, 20s integration time, room temperature). Samples were kept immersed in buffer during SERS measurements.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b06007. Additional information on sensor saturation conditions and performance in fetal calf serum. (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported in part by the National Basic Research Program of China (no. 2011CB933101, 2015CB932004), National Natural Science Foundation of China (grant no. 21303208). The Project was also sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (no. Y31Z55). The StartUp Funding from the Institute of High Energy Physics of the Chinese Academy of Sciences (no. 2011IHEPYJRC504) is also gratefully acknowledged. J.A.H. acknowledges the support of the Australian Research Council DECRA scheme (DE130101300) and the infrastructure support from the Australian Research Council Grant LF 100100117. REFERENCES (1) International Agency for Research on Cancer. GLOBOCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012; IARC: Lyon, France 2013; http://globocan.iarc.fr/ (accessed May, 2015). (2) Arzumanyan, A.; Reis, H. M.; Feitelson, M. A. Pathogenic Mechanisms in HBV- and HCV-Associated Hepatocellular Carcinoma. Nat. Rev. Cancer 2013, 13, 123−135. (3) Australian Institute of Health and Welfare. Cancer Incidence Projections: Australia, 2011 to 2020; Cancer Series no. 66, Cat. No. CAN 62; AIHW; Canberra, Australia; 2012; http://www.aihw.gov.au (accessed May, 2015). (4) Yu, C.; Qian, L.; Uttamchandani, M.; Li, L.; Yao, S. Q. SingleVehicular Delivery of Antagomir and Small Molecules to Inhibit miR122 Function in Hepatocellular Carcinoma Cells by using “Smart” H

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano Mesoporous Silica Nanoparticles. Angew. Chem., Int. Ed. 2015, 54, 10574−10578. (5) World Gastroenterology Organisation Global Guidelines: Hepatocellular Carcinoma (HCC): A Global Perspective; World Gastroenterology Organisation: Milwaukee, WI, 2009; www.worldgastroenterology.org (accessed May, 2015). (6) Xu, L.; Hazard, F. K.; Zmoos, A. F.; Jahchan, N.; Chaib, H.; Garfin, P. M.; Rangaswami, A.; Snyder, M. P.; Sage, J. Genomic Analysis of Fibrolamellar Hepatocellular Carcinoma. Hum. Mol. Genet. 2015, 24, 50−63. (7) Wu, L. J.; Pan, Y. D.; Pei, X. Y.; Chen, H.; Nguyen, S.; Kashyap, A.; Liu, J.; Wu, J. Capturing Circulation Tumor Cells from Hepatocellular Carcinoma. Cancer Lett. 2012, 326, 17−22. (8) Wu, L.; Qu, X. Cancer Biomarker Detection: Recent Achievements and Challenges. Chem. Soc. Rev. 2015, 44, 2963−2997. (9) Lok, A. S.; Sterling, R. K.; Everhart, J. E.; Wright, E. C.; Hoefs, J. C.; Di Bisceglie, A. M.; Morgan, T. R.; Kim, H. Y.; Lee, W. M.; Bonkovsky, H. L.; Dienstag, J. L. Des-γ-Carboxy Prothrombin and AlphaFetoprotein as Biomarkers For the Early Detection of Hepatocellular Carcinoma. Gastroenterology 2010, 138, 493−502. (10) Dinish, U. S.; Balasundaram, G.; Chang, Y. T.; OLivo, M. Sensitive Multiplex Detection of Serological Liver Cancer Biomarkers Using SERS-Active Photonic Crystal Fiber Probe. J. Biophotonics 2014, 7, 956−965. (11) Yu, J. P.; Xu, X. G.; Ma, R. J.; Qin, S. N.; Wang, C. R.; Wang, X. B.; Li, M.; Li, M. S.; Ma, Q.; Xu, W. W. Development of a Clinical Chemiluminescent Immunoassay for Serum GPC3 and Simultaneous Measurements Alone With AFP and CK19 in Diagnosis of Hepatocellular Carcinoma. J. Clin. Lab. Anal. 2015, 29, 85−93. (12) Wang, Y. C.; Yang, H. Y.; Xu, H. F.; Lu, X.; Sang, X. T.; Zhong, S. X.; Huang, J. F.; Mao, Y. L. Golgi Protein 73, not Glypican-3, May Be a Yumor Marker Complementary to Alpha-Fetoprotein for Hepatocellular Carcinoma Diagnosis. J. Gastroenterol. Hepatol. 2014, 29, 597−602. (13) Marrero, J. A.; Feng, Z.; Wang, Y.; Nguyen, M. H.; Befeler, A. S.; Roberts, L. R.; Reddy, K. R.; Harnois, D.; Llovet, J. M.; Normolle, D.; Dalhgren, J.; Chia, D.; Lok, A. S.; Wagner, P. D.; Srivastava, S.; Schwartz, M. α-Fetoprotein, Des-γ Carboxyprothrombin, and Lectin-Bound αFetoprotein in Early Hepatocellular Carcinoma. Gastroenterology 2009, 137, 110−118. (14) Biselli, M.; Conti, F.; Gramenzi, A.; Frigerio, M.; Cucchetti, A.; Fatti, G.; D’Angelo, M.; Dall’Agata, M.; Giannini, E. G.; Farinati, F. A New Approach to The Use of Alpha-Fetoprotein as Surveillance Test for Hepatocellular Carcinoma in Patients With Cirrhosis. Br. J. Cancer 2015, 112, 69−76. (15) Capurro, M.; Wanless, I. R.; Sherman, M.; Deboer, G.; Shi, W.; Miyoshi, E.; Filmus, J. Glypican-3: A Novel Serum and Histochemical Marker for Hepatocellular Carcinoma. Gastroenterology 2003, 125, 89− 97. (16) Le Ru, E.; Etchegoin, P. Principles of Surface-Enhanced Raman Spectroscopy and related plasmonic effects, 1st ed.; Elsevier: Amsterdam, 2008. (17) Porter, M. D.; Lipert, R. J.; Siperko, L. M.; Wang, G.; Narayanan, R. SERS As a Bioassay Platform: Fundamentals, Design, and Applications. Chem. Soc. Rev. 2008, 37, 1001−1011. (18) Wang, G.; Lipert, R. J.; Jain, M.; Kaur, S.; Chakraboty, S.; Torres, M. P.; Batra, S. K.; Brand, R. E.; Porter, M. D. Detection of The Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering. Anal. Chem. 2011, 83, 2554−2561. (19) Granger, J. H.; Granger, M. C.; Firpo, M. A.; Mulvihill, S. J.; Porter, M. D. Toward Development of A Surface-Enhanced Raman Scattering (SERS)-Based Cancer Diagnostic Immunoassay Panel. Analyst 2013, 138, 410−416. (20) Harper, M. M.; McKeating, K. S.; Faulds, K. Recent Developments and Future Directions in SERS for Bioanalysis. Phys. Chem. Chem. Phys. 2013, 15, 5312−5328. (21) Li, M.; Cushing, S. K.; Zhang, J.; Suri, S.; Evans, R.; Petros, W. P.; Gibson, L. F.; Ma, D.; Liu, Y.; Wu, N. Three-Dimensional Hierarchical Plasmonic Nano-Architecture Enhanced Surface-Enhanced Raman

Scattering Immunosensor for Cancer Biomarker Detection in Blood Plasma. ACS Nano 2013, 7, 4967−4976. (22) Wang, Y.; Vaidyanathan, R.; Shiddiky, M. J. A.; Trau, M. Enabling Rapid and Specific Surface-Enhanced Raman Scattering Immunoassay Using Nanoscaled Surface Shear Forces. ACS Nano 2015, 9, 6354− 6362. (23) Devi, R. V.; Doble, M.; Verma, R. S. Nanomaterials for Early Detection of Cancer Biomarker with Special Emphasis on Gold Nanoparticles in Immunoassays/Sensors. Biosens. Bioelectron. 2015, 68, 688−698. (24) Cao, Y. C.; Jin, R. C.; Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Raman Dye-Labeled Nanoparticle Probes for Proteins. J. Am. Chem. Soc. 2003, 125, 14676−14677. (25) Guerrini, L.; Krpetić, Ž .; van Lierop, D.; Alvarez-Puebla, R. A.; Graham, D. Direct Surface-Enhanced Raman Scattering Analysis of DNA Duplexes. Angew. Chem., Int. Ed. 2015, 54, 1144−1148. (26) Ye, J.; Chen, Y.; Liu, Z. A Boronate Affinity Sandwich Assay: An Appealing Alternative to Immunoassays for the Determination of Glycoproteins. Angew. Chem., Int. Ed. 2014, 53, 10386−10389. (27) Cui, Y.; Ren, B.; Yao, J. L.; Gu, R. A.; Tian, Z. Q. Multianalyte Immunoassay Based on Surface-Enhanced Raman Spectroscopy. J. Raman Spectrosc. 2007, 38, 896−902. (28) Wang, G.; Park, H. Y.; Lipert, R. J.; Porter, M. D. Mixed Monolayers on Gold Nanoparticle Labels for Multiplexed SurfaceEnhanced Raman Scattering Based Immunoassays. Anal. Chem. 2009, 81, 9643−9650. (29) Wang, Y.; Rauf, S.; Grewal, Y. S.; Spadafora, L. J.; Shiddiky, M. J. A.; Cangelosi, G. A.; Schlucker, S.; Trau, M. Duplex Microfluidic SERS Detection of Pathogen Antigens with Nanoyeast Single-Chain Variable Fragments. Anal. Chem. 2014, 86, 9930−9938. (30) Kho, K. W.; Dinish, U. S.; Kumar, A.; Olivo, M. Frequency Shifts in SERS for Biosensing. ACS Nano 2012, 6, 4892−4902. (31) Guerrini, L.; Pazos, E.; Penas, C.; Vázquez, M. E.; Mascareñas, J. L.; Alvarez-Puebla, R. A. Highly Sensitive SERS Quantification of the Oncogenic Protein c-Jun in Cellular Extracts. J. Am. Chem. Soc. 2013, 135, 10314−10317. (32) Wang, Y.; Ji, W.; Sui, H.; Kitahama, Y.; Ruan, W.; Ozaki, Y.; Zhao, B. Exploring the Effect of Intermolecular H-Bonding: A Study on Charge-Transfer Contribution to Surface-Enhanced Raman Scattering of p-Mercaptobenzoic Acid. J. Phys. Chem. C 2014, 118, 10191−10197. (33) Wang, Y.; Yu, Z.; Ji, W.; Tanaka, Y.; Sui, H. M.; Zhao, B.; Ozaki, Y. Enantioselective Discrimination of Alcohols by Hydrogen Bonding: A SERS Study. Angew. Chem., Int. Ed. 2014, 53, 13866−13870. (34) Perumal, J.; Kong, K. V.; Dinish, U. S.; Bakker, R. M.; Olivo, M. Design and Fabrication of Random Silver Films as Substrate for SERS Based Nano-Stress Sensing of Proteins. RSC Adv. 2014, 4, 12995− 13000. (35) Grubisha, D. S.; Lipert, R. J.; Park, H. Y.; Driskell, J.; Porter, M. D. Femtomolar Detection of Prostate-Specific Antigen: An Immunoassay Based on Surface-Enhanced Raman Scattering and Immunogold Labels. Anal. Chem. 2003, 75, 5936−5943. (36) Xia, Y.; Whitesides, G. M. Soft Lithography. Angew. Chem., Int. Ed. 1998, 37, 550−575. (37) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Soft Lithography in Biology and Biochemistry. Annu. Rev. Biomed. Eng. 2001, 3, 335−373. (38) Li, H.; Zhang, J.; Zhou, X.; Lu, G.; Yin, Z.; Li, G.; Wu, T.; Boey, F.; Venkatraman, S. S.; Zhang, H. Aminosilane Micropatterns on HydroxylTerminated Substrates: Fabrication and Applications. Langmuir 2010, 26, 5603−5609.

I

DOI: 10.1021/acsnano.5b06007 ACS Nano XXXX, XXX, XXX−XXX