Ultrasensitive detection of protein with wide linear ... - ACS Publications

Southeast University, Nanjing 210096, China. §. State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engin...
0 downloads 0 Views 1MB Size
Download PDF
Subscriber access provided by UNIVERSITY OF CONNECTICUT

Article

Ultrasensitive detection of protein with wide linear dynamic range based on core-shell SERS nanotags and photonic crystal beads Bing Liu, Haibin Ni, Di Zhang, Delong Wang, Degang Fu, Hong-Yuan Chen, Zhongze Gu, and Xiang-Wei Zhao ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00310 • Publication Date (Web): 06 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sensors is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

Ultrasensitive detection of protein with wide linear dynamic range based on core-shell SERS nanotags and photonic crystal beads Bing Liu,†,‡ Haibin Ni,†,‡ Di Zhang,†,‡ Delong Wang,†,‡ Degang Fu,†,‡ Hongyuan Chen,§ Zhongze Gu†, ‡ and Xiangwei Zhao*,†, ‡ †

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China ‡ National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing 210096, China § State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

ABSTRACT: Detection of proteins in a wide concentration range from fg mL-1 to sub mg mL-1 is a challenge in the high throughput analysis of precision medicine. Herein, we proposed a biosensor consisted of core-shell surface-enhanced Raman scattering (SERS) nanotags as labels and photonic crystal beads (PCBs) as carriers for ultrasensitive detection of proteins. In practice, Raman Dyes (RDs) were embedded in the interface of gold core and silver shell in the bimetal nanoparticles to form SERS nanotags. It was found that the sensitivity was significantly improved due to the enhanced Raman signal by the coupling of the core-shell structure and linear dynamic range (LDR) was extended owing to the high surface to volume ratio of PCBs as well. In addition, we also demonstrated that the biosensor exhibited fine stability and low background, which has great application potential in the detection of protein biomarkers. KEYWORDS: gold-silver core-shell nanoparticle, SERS nanotags, photonic crystal, ultrasensitive detection, linear dynamic range As an emerging approach for disease treatment and prevention, precision medicine takes into account individual variability in genes, environment and lifestyle for each person, which will greatly improve our understanding of disease and help to develop personalized medical programs. It is necessary to incorporate new technological advances, such as genomics, proteomics and metabolomics so that huge biomolecular information will be obtained and analyzed.1 This is especially important for the protein marker discovery and validation.2 However, to detection of panels of protein biomarkers is still a challenge due to the complex sample and it’s pretreatment. Another reason is that the concentrations of the protein needed to be accurately measured also differ in the range from fg mL-1 to sub mg mL-1 among different samples.3, 4 For example, C reactive protein (CRP) is a marker for both cardiovascular disease and inflammation. However, the clinical range of CRP concentration used for

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the prediction of future heart-related disease is at least ten times lower than that in used as a marker for inflammation.5 In addition, the CRP concentration in human plasma spans almost 6 orders of magnitude. In the quantitative detection of CRP, series of sample dilution are always needed to fit the linear dynamic range (LDR). Therefore, protein detection methods with both high sensitivity and wide LDR are much more and more desired for the fast-developing technologies in precision medicine. Usually the detection of protein marker is based on heterogeneous immunoassays, such as colorimetric assay,6 enzyme-linked immunosorbent assay (ELISA),7 chemiluminescent,8 fluorescent9 and electrochemical immunoassay.10 In these immunoassays, the target protein or antigen is sandwiched between capture antibody immobilized on a solid carrier and detection antibody linked with a label. Generally, the sensitivity or the limit of detection (LOD) is determined by the signal intensity of label measured under certain signal-to-noise ratio, and the LDR mainly depends on the surface to volume ratio of the solid carrier. Up to now, lots of attempts have been made to increase the sensitivity with nanomaterial as labels or enlarge the LDR with porous materials as the solid carriers,11, 12 However, analytical methods both fulfill them are seldom reported with respect to the problems of protein detection in the precision medicine. Recently, surface-enhanced Raman scattering (SERS) becomes one of the most widely studied analytical techniques in the field of biochemistry, biomedicine, food safety and environment monitoring due to its high sensitivity, spectral specificity, stability and multiplex capability.12-17 Raman Dyes (RDs) with large Raman scattering cross sections are good candidates as labels to gain amplified signal when they are absorbed on surface of noble metal nanoparticles like gold or silver, which usually has SERS enhancement factor of about 106. The sensitivity of detection based on the so-called SERS nanotag could be down to single-molecule if their structures are rationally designed. For example, Lim et al embedded RDs in the interior gaps of core-shell gold nanoparticles which have higher enhancement factors than commonly used gold nanoparticles and found that the sensitivity was sufficient for single-molecule detection.18 Except for that, core-shell SERS nanotags have uniform and narrow distribution of “hotspots”, which is also important for reproducible ultrasensitive bioassay.19 In addition, compared with fluorescence dyes, SERS nanotags are more tolerant to photo bleaching and quenching. All these characters make SERS nanotag more and more popular both for in vitro diagnostics and in vivo analysis.12, 13, 20 In terms of SERS nanotags, the enhancement factor of usually used gold nanoparticles (AuNPs) is 100-1,000 times lower than that of silver nanoparticles (AgNPs) owing to the fact that the extinction coefficient of the surface plasmon band of AgNP is approximately 4 times as compared to the same size AuNP.21, 22 However, it is hard to control the size of AgNP in synthesis, which will destroy the reproducibility and consistency of assays. Therefore, embedding the RDs in the bimetal interface of gold-silver core-shell nanoparticles (Au@Ag NPs) will combine both the advantages of high SERS enhancement and uniform sizes.23-25

ACS Paragon Plus Environment

Page 2 of 18

Page 3 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

Figure 1. Schematic illustration of protein detection by SERS nanotag of Au@Ag NP and PCB.

Photonic crystal beads (PCBs), which are self-assembled from monodispersed colloidal nanoparticles, have been proved to be a sensitive carrier with high surface to volume ratio because their surfaces have ordered roughness in the scale of several hundreds of nanometers.26-28 Hence, in this paper, we proposed an ultrasensitive biosensor based on gold-silver core-shell SERS nanotags and PCBs for the detection of protein with wide concentration range. The principle is illustrated in Figure 1, first we synthesized the SERS nanotags by absorbing RDs onto the surface of AuNPs, following by synthesized silver shell and modified with PEG and detection antibodies. Then, capture antibodies were immobilized on PCBs as solid immunoassay carriers. After they formed immunocomplex with the analytes, Raman spectra were measured and the analytes could be quantitative detected by Raman signal intensities of the SERS nanotags. Our results showed that the sensitivity and LDR of this biosensor for CRP were 478 fg mL-1 and from 5 pg mL-1 to 10 µg mL-1 respectively, which also holds promise for the detection of other protein biomarkers.

EXPERIMENTAL SECTION Materials All distilled water was purified to Millipore Milli-Q quality. Glasswares were first soaked in freshly prepared aqua regia solution (HCl/HNO3, 3:1) overnight, and subsequently rinsed thoroughly with water before use. Trisodium citrate (Na3C6H5O7), hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O), bovine serum albumin (BSA) and (3-Glycidoxypropyl)-trimethoxysilane (GPTMS, 98 %) were obtained from Sigma-Aldrich. Thiolated PEG (PEG-SH, MW ~ 5 kDa) and thiolated-carboxylated PEG (HS-PEG-COOH, MW ~ 5 kDa) were commercially purchased from Laysan Bio, Inc. (USA). Nile blue A (NBA), ethyl dimethylaminopropyl carbodiimide (EDC), silver nitrate (AgNO3), ascorbic acid and sulfo-N-hydroxysuccinimide (NHS) were purchased from Alfa Aesar. All reagents were used without further purification. Human IgG, mouse IgG, anti-human IgG and anti-mouse IgG functional fragments were received from Biodee Biotechnology Co., Ltd. (China). Recombinant human CRP and mouse anti-human CRP monoclonal antibody were obtained from Abcam. Human AFP, CEA and CA125, a pair of mouse anti-human AFP monoclonal antibodies, a pair of mouse anti-human CEA monoclonal antibodies and a pair of

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mouse anti-human CA125 monoclonal antibodies were commercially purchase from Beijing Key-bio Biotech Co., Ltd (China). Monodisperse colloidal silica nanoparticles ranging from 186 to 315 nm in diameter were purchased from Nanjing Nanorainbow Biotechnolgoy Co., Ltd. (China). Phosphate-buffered saline (PBS, 0.05 M, and pH = 7.4) was prepared in-house. Preparation of photonic crystal bead carriers The fabrication of the silica photonic crystal beads was based on the method of co-flow microfluidic device, which was described by Zhao et al. (Supporting Information).29 Subsequently, the capture antibodies were immobilized on the silica PCB as follows. First, the silica PCBs were completely immersed in piranha solution (H2O2/H2SO4, 3:7) for 12 h. Then these PCBs were thoroughly washed with deionized water, and were dried under a stream of nitrogen. Next, GPTMS (5 %, in toluene) was mixed with the PCBs overnight at room temperature (25 ℃) for salinization.30 Then, nonspecific binding GPTMS on the surface of the PCBs were removed by rinsing several times with toluene and ethanol, and also dried under nitrogen. Active epoxy groups were exposed for conjugating capture antibody functional fragments on the surface of the nanostructures of the PCBs, and then the PCBs were incubated with 1 mg/mL functional fragments at 4 ℃ for 12 h. Next, 0.5 % BSA solution was added to the PCBs for blocking non-reaction site for 2 h at room temperature and washed with PBS several times. Finally, the PCBs were stored at 4 ℃ for further use. Synthesis of gold-silver core-shell nanoparticles Gold nanoparticles were fabricated by following the well-known Frens method31 and used as first substrates for the SERS nanotags preparation. Briefly, 100 mL HAuCl4 (0.01 %, w/w) solution was heated to boiling. Next, 0.75 mL of 1 % (w/w) sodium citrate solution was rapidly added under vigorous stirring. After the mixture had boiled for 10 min, heating was stopped until the color did not change. The solution was cooled down to room temperature with continuous stirring. The preparation of Au@NBA@Ag NPs was adapted from reference,32 first of all, the NBA molecules were directly adsorbed onto the surface of AuNPs by the electrostatic interaction. 5 mL different concentrations of NBA solutions (0, 0.2…2.4 µM) were added to the colloidal gold (45 mL) and the mixture was allowed to react 20 min under vigorous stirring, respectively. Then the NBA modified AuNPs (Au@NBA NPs) were centrifuged three times to remove the unabsorbed NBA, and re-suspended in water (10 mL). Afterwards, 2 mL of 0.1 M ascorbic acid was added to the Au@NBA NPs solution in a beaker under magnetic stirring. Next, 1 mM AgNO3 was dropwise added into the above mixtures, and the finally concentrations of AgNO3 solutions were 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 µM. Owing to the reduction of AgNO3 by ascorbic acid and the resultant silver shells continuously grew on the surface of the AuNPs. When the color of the solution changed from wine red to orange yellow, the solutions continued to stir for 30 min and centrifuged three times at 10,000 rpm for 10 min. After removal of the supernatant, the Au@NBA@Ag NPs

ACS Paragon Plus Environment

Page 4 of 18

Page 5 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

were obtained. Preparation of SERS nanotag HS-PEG-COOH (10 µM, 1.5 mL) was added to the Au@NBA@Ag NPs solution and mixed for 20 min under stirring. Subsequently, PEG-SH (50 µM, 10 mL) was added to the mixture and further incubated for 3 h. The resulting Au@NBA@Ag@PEG NPs were centrifuged three times at 10,000 rpm for 10 min to remove excess PEG and re-suspended in 2 mL PBS. Afterwards, EDC and NHS were used to activate carboxyl groups of PEG for effective antibodies bioconjugation by covalent bonding. Freshly prepared EDC (40 mg/mL, 12 µL) and NHS (110 mg/mL, 12 µL) were added to the PEG encapsulated nanoparticles and mixed vigorously for 20 min at 25 ℃. The excess EDC and NHS were eliminated by centrifugation and the sediments were re-suspended in 2 mL PBS. Subsequently, detection antibody functional fragments (100 µL, 80 µg/mL) were added to the activated nanoparticles and incubated for 2 h at 25 ℃ and then kept overnight at 4 ℃. After being centrifuged three times, the final SERS nanotag was obtained and stored at 4 ℃ for further use. Preparation and evaluation of the biosensor For evaluating the biosensor, the analysis of different concentrations of mouse IgG ranging from 1 pg mL-1 to 100 µg mL-1 was performed. Silica PCB immobilized with capture antibodies were incubated with mouse IgG (10 µL/bead) in test tubes for 30 min at 37 ℃ under shaking. In order to remove nonhybridized mouse IgG, the silica PCBs were washed five times with PBS. Next, the silica PCBs were mixed with SERS nanotags and incubated for 30 min under the same conditions. Subsequently, the nonspecific adsorption SERS nanotags were also eliminated by rinsing thoroughly with PBS. Finally, Raman signal of SERS nanotags on the silica PCBs were measured using a Raman spectrometer coupled to a microscope for the quantitative analysis of mouse IgG. C-reactive protein analysis Sample solutions with CRP concentrations in the range of 500 fg mL-1 to 100 µg mL-1 were incubated with the silica PCBs at 37 ℃ were incubated under stirring. After rinsing thoroughly with PBS, SERS nanotags were added to the silica PCBs and incubated for 30 min at 37 ℃. Next, the silica PCBs were washed with PBS five times. Afterwards, Raman signal of SERS nanotags on the silica PCBs were recorded by a Raman spectrometer coupled to a microscope. For selectivity evaluation, the concentrations of CRP, AFP, CEA, CA125, human IgG and mouse IgG were set to 100 ng mL-1. The reaction and analysis procedures were as same as those of CRP analysis. For clinical sample analysis, five serum samples were obtained from Zhongda Hospital affiliated to Southeast University. The CRP concentrations of these samples were tested with as-proposed biosensor and commercialized hs-CRP ELISA test kit from MP Biomedicals (Fisher Scientific, UK) according to the manual respectively.

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Electromagnetic simulations FDTD simulation was performed using a commercial software (FDTD Solutions, Lumerical Inc.). The models in the simulation are shown in Figure S-1 (Supporting Information). Four interior nanobridges were added between gold core and silver shell in a symmetrical position because the formation of silver shell was supported by gold core with the same crystalline lattice constant.33 And we assumed that the NBA molecules formed monolayer on the surface of AuNP, and its thickness is calculated about 0.8 nm.34 For Au@Ag NP, the diameter of core was 60 nm, the dimensions of each nanobridge were 2.5 nm × 0.8 nm (cylindrical shape), the gap size was 0.8 nm, and shell thicknesses of silver and PEG layer were 6 and 3 nm, respectively. For AuNP, the core diameter, the thickness of NBA molecules layer and PEG shell were as same as those of Au@Ag NP, respectively. The refractive indices of NBA monolayer and PEG were 1.332 and 1.469, respectively (Supplementary Information). A linearly polarized plane wave (λ= 785 nm) incident along the z-axis was used for plasmon excitation. Instrumentation The reflection and Raman spectra were acquired by an optical fiber spectrometer (Ocean Optics, QE65000) and Raman spectrometer (PeakSeeker Pro 785E, Oceanoptics, USA) coupled to a microscope (Olympus, BX51) with a 50× objective. The Raman excitation source used in whole bioassay was 785 nm laser with a power of 5 mW and a typical integration time of 10 s. For each sample measurement, five independent silica PCBs were measured. Scanning electron microscopy (SEM; Zeiss, Ultra Plus) and transmission electron microscopy (TEM; JEOL, JEM-2100) were applied to characterize structure and size of the PCBs and SERS nanotags. The UV-vis adsorption spectra were acquired by using a Hitachi 5000 UV/Vis/NIR spectrophotometer.

RESULTS AND DISCUSSION Synthesis of gold-silver core-shell nanoparticles In our biosensor, the SERS nanotag acts as label for quantitative analysis. Therefore, the Raman signal of the SERS nanotag must be optimized for high intensity and reproducibility. Electromagnetic field enhancement derived from localized surface plasmon resonance (LSPR) contributes mainly for the exponentially enhanced Raman signal of the RDs in the Au@Ag NPs,35 whose structure is schematically illustrated in Figure 1. On the one hand, the silver shell of Au@Ag NP has a high degree of plasmonic tenability.36 When RDs have been embedded into interface of bimetal, the hybridization between inner gold nanosphere and the outer silver shell makes the LSPR more greatly depend on the geometrical factors such as core diameter, thickness of RD layer and outer shell.37 We first choose the size of the gold core as 60 nm because it has better SERS enhancement than others.38 Also, the nanoparticle size is larger than that of pores between nanoparticles on the surface of PCBs (Figure S-2 in

ACS Paragon Plus Environment

Page 6 of 18

Page 7 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

the Supporting Information) so that they could not enter the interior of PCBs and the background signal will not be increased. Figure 2 shows the size of AuNPs we synthesized is 60.24 ± 2.18 nm and the monodispersity is 3.6 %, which is fine enough for the synthesis of Au@Ag NP with reproducible Raman signal. Then we used these AuNPs as cores for SERS nanotags in the following experiments.

Figure 2. TEM of AuNPs (a) and their size distribution (b).

On the other hand, due to the ultrahigh enhancement of gap mode electromagnetic field, the number of RDs, which embedded into the interface of bimetal would greatly affect the intensity of Raman signal of SERS nanotag. More RDs will result in higher SERS signal. However, excess RDs will lead to the aggregation of AuNPs in the synthesis because they neutralize the surface charge of AuNPs, which will decrease the monodispersity of the nanoparticles. In order to determine the best RD concentration for the maximum Raman enhancement, different concentrations of NBA were absorbed onto the surface of AuNPs by electrostatic interaction (Au@NBA NPs). Figure 3a shows the UV-vis absorption spectra of these functional nanoparticles, it is clear that the position of the absorption peaks shifted from 530 to 545 nm upon the absorption of NBA when the concentrations ranged from 0.2 to 2.2 µM, this is consistent with conclusion that the surface plasmon band of AuNPs will red-shift upon their surfaces modified with moieties.39 In addition, the full width at half wavelength (FWHW) of absorption peak changed wide when the concentration of NBA was 2.4 µM, indicating the formation of nanoparticles aggregation. This is due to the neutralization of the AuNPs surface charge when the amount of NBA absorbed was increased. Raman spectra of these nanoparticles were shown in Figure 3b, along with the increase of the concentration of NBA absorbed onto the surface of AuNPs, the Raman intensity was significantly increased, which indicated the function of AuNPs as optical enhance agents. Because of the nice monodispersity of Au@NBA NPs and the strong enough SERS signal of NBA at 2.0 µM, this concentration was chosen for the next step.

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. UV-vis absorption spectra (a) and Raman spectra (b) of Au@NBA NPs as a function of NBA concentrations.

In order to determine the best nanoparticle size for the maximum Raman enhancement, we also prepared Au@NBA@Ag NPs with different thicknesses of silver shells and investigated the effects of shell thickness on SERS signal.40 The silver metal could be selectively grown around gold core to form the core-shell NP with embedded NBA because of the match of crystalline lattice between silver and gold.33 As shown in Figure 4a, the color of the newly formed nanoparticles colloidal solutions change from amaranth to aurantiacus with the increase of the concentration of AgNO3 (from 0 up to 4 µM) added to the Au@NBA NPs, which demonstrated that the thicknesses of silver shell of the core-shell NPs gradually rises with the increase of the amount of AgNO3. At the same time, the position of the absorption peak of LSPR of the nanoparticles blue-shifted from 530 to 500 nm (Figure 4b), which fits well with the traditional Mie scattering theory and the support of the dielectric data.41-43 The appearance of LSPR band of the silver shell at 397 nm also indicates that the shell thickness is increasing. We then investigated the intensity of their Raman signal as shown in Figure 4c. It comes out that the Raman signal intensity of Au@NBA@Ag NPs is strongly related to the concentration of AgNO3 and hence the silver thickness, but not in a linear relationship. The thickness of silver shell is about 6 nm when the concentration of AgNO3 is 2.5 µM (Figure 4d). According to the theoretical calculation, the silver shell thickness of core-shell NPs changes approximately from 1.38 to 8.78 nm with the increasing addition of AgNO3 (0-4.0 µM). Compared with the bare AuNPs, the Raman signal intensity increases gradually when the concentration of AgNO3 is increased from 0 to 2.5 µM (the silver shell thickness is about from 1.38 to 6.0 nm) and then declines when higher concentration (3.0-4.0 µM) (the thickness of silver shell is increased from 6.96 to 8.78 nm) is used. The hybridization of the plasmons of the gold core and silver shell will lead to split bonding and antibonding modes and the “hotspots” are mostly confined in the gap of core and shell when the thickness is relatively thin.44-46 However, when the shell thickness is too large, the coupling of them will become weak and then the “hotspots” will transfer to the outer surface of the shell. Besides, thick silver shell also prevents

ACS Paragon Plus Environment

Page 8 of 18

Page 9 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

the output of the Raman signal of RDs. Therefore, in this paper, we chose 2.5 µM AgNO3 for the Au@NBA@Ag NPs preparation to get better signal intensity for further experiments

Figure 4. Color changes of Au@NBA@Ag NPs with different Ag shell thicknesses (a), UV-vis absorption (b) and Raman spectra (c) of Au@NBA@Ag NPs as a function of AgNO3 concentrations. (d) TEM image of core-shell nanoparticles. Inset is high magnification image of nanoparticle.

Modification of core-shell SERS nanotag The aggregation of SERS nanotags will affect the assay reproducibility although it may result in much more and stronger extra “hotspots” for greater enhancement. In order to prevent the aggregation of nanoparticles during storage and analysis, it is necessary to modify their surface with protection layer of polymer such as PEG.47, 48 Therefore, in this paper, mixture of HS-PEG-COOH and PEG-SH was used to encapsulate the surface of Au@NBA@Ag NPs with the former as a linker for antibody conjugation and the latter to reduce the steric effect. Then, UV-vis absorption spectra were used to confirm the conjugation of antibody on the surface of Au@NBA@Ag NPs and monodispersity of SERS nanotags. From Figure 5a, it could be seen that the characteristic absorption peak of AuNPs with a diameter of about 60 nm is 530 nm, and it shifts to 536 nm after RDs absorption. However, after the silver shell fabrication, the peak shifts to 504 nm, and the characteristic absorption peak of silver shell at 397 nm appears. Subsequently, after PEG encapsulation and detection antibody conjugation, the peak of AuNPs and silver shell shift to 508 nm, 516 nm and 400 nm, 404 nm, respectively. In addition, the FWHWs of the absorption peaks of AuNPs are not significantly changed and the absorption spectra are symmetric, which indicate that the core-shell nanotags maintain a nice monodispersity and uniformity.

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

We also checked the Raman spectra after every steps of SERS nanotag synthesis. It was founded that the SERS background of the PEG is ignorable (Figure 5b) despite its thickness is 3 nm (Figure 4d) and the Raman intensity of Au@NBA NP is enhanced by about 19 times after the formation of Ag shell, which is valuable for the achievement of high signal-to-noise ratio. Besides the low Raman scattering of PEG, another reason may be that the enhanced “hotspots” is mainly confined in the gap of core and shell where the RDs are embedded (Figure 5c). Hence, the core-shell nanostructure of the as-prepared Au@NBA@Ag nanotag improved the signal-to-noise ratio from view point of the label in assays. Therefore, the core-shell nanotag not only have amplified signal intensity but also have better stability, which is favored by reproducible and highly sensitive assays.

Figure 5. UV-vis absorption spectra (a) and Raman spectra (b) of SERS nanotags at different synthesis steps. Calculated near-field electromagnetic field distributions of the Au@Ag NP (c) and AuNP (d) by finite difference time domain-based electromagnetic simulation. (e) Comparison of the line electromagnetic field distribution profile along the maximum point-line (white dotted line) at an incident wavelength of 785 nm.

Evaluation of the biosensor In order to evaluate the quantitative performance of the biosensor by Au@NBA@Ag NPs-based SERS nanotags and silica PCBs, a quantitative assay was performed for protein detection. Here, PCBs with a reflection peak at 583 nm were used as reaction carriers for the detection of mouse IgG in a sandwich immunoassay. The PCBs have mechanically and chemically stable with extremely low Raman backgrounds because they are composed of silica nanoparticles connected to each other by sintering (Figure S-3 in the Supporting Information).29 Therefore, when the PCBs are used as carriers, the Raman background will not interfere with the Raman signal from the SERS nanotags as labels, which benefits the improvement of

ACS Paragon Plus Environment

Page 10 of 18

Page 11 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

signal-to-noise ratio from point view of carrier. What’s more, the fingerprinting molecular vibrational spectra of RDs embedded in the gap of the bimetal are intrinsically much more stable, which is desirable for improved assay robustness and reproducibility. After the sandwich immunoassays, the intensities of Raman shift of the SERS nanotag at 595 cm-1 are plotted against the concentration of mouse IgG, as shown in Figure 6. The concentrations we used range from 1 pg mL-1 to 100 µg mL-1, spanning 8 orders of magnitudes. It could be seen that the dose response is pretty good. In the meantime, the limit of detection and the limit of quantity (LOQ) are 672 fg mL-1 and 2.32 pg mL-1 based on signal-to-noise of 3:1 and 10:1, respectively (Figure 6b and inset). In comparison with previously reported detection method based on AuNPs-based SERS nanotags and PCBs the LOD is improved by 14 times for mouse IgG.27 Here, the linear dynamic detection range is from 10 pg mL-1 up to 10 µg mL-1 (R2 = 0.945), spanning 6 orders of magnitudes, which is also wider than those of the commonly used method of ELISA and other detection methods.49, 50 PCBs we used here are composed of 253 nm silica nanoparticles connected to each other by sintering. The pore size of PCBs is less than 38 nm (15 % of the nanoparticle diameter) and much less than the diameter of core-shell SERS nanotag (about 78 nm). Figure 6c shows the SERS nanotags on the surface of PCB after immunoreaction. Therefore, the nanotags could not enter the pores of PCBs and the washing of nonspecific absorbed nanotags will be facilitated. Our results agree well that the background does not decrease anymore just after 3 to 4 rounds of washing with PBS solution (Figure S-4 in the Supporting Information). Figure 6c also shows clearly that the surface of the PCB is composed of nanoparticles, which greatly increases the surface area and will benefit wide LDR. In addition, not like the usually used porous carriers or substrates, the pores here is immune to larger sized SERS nanotags. Hence, the noise from nonspecific absorption is greatly reduced, which also favors high signal-to-noise ratio and strengths the reasonability of the combination of PCBs and SERS nanotags.

Figure 6. Quantitative detection of mouse IgG with SERS nanotags and PCBs. (a) Raman spectra of different concentrations of mouse IgG (from 1 pg mL-1 to 100 µg mL-1). (b) Reference plot of Raman intensities at Raman shift 595 cm-1 vs. logarithm of mouse IgG concentrations. Error bar is calculated from five repeats. Inset is the linear part of the reference plot. (c) High magnification image of SERS nanotags on PCB surface after immunoreaction when the mouse IgG concentration is 10 pg/mL.

From the simulation results as shown in Figure 5e, it could be seen that electromagnetic enhancement is highly localized in the interior gap region of the

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Au@Ag NP, and the magnitude of the maximal enhanced electric field is 42.3 times higher than that of the incident light. However, in comparison, the maximum enhancement obtained from AuNP is only 9.3 times. Because the SERS signals are proportional to the biquadrate of the electric field (|E|4), the enhancement factor of core-shell SERS nanotag will be more than 256 times of that of AuNPs. All of these enhancement effects are valuable for ultrasensitive analysis. Another point need to be mentioned is that the photonic crystal structure could also enhance the SERS signal as well.51, 52 Also, the coefficient of variation of the assay among different batches is as low as 5.8 %, which illustrates the advantages of biosensor in terms of uniformity, stability and background signals. C-reactive protein analysis To test whether the as-developed biosensor is suitable for the reliability detection of protein markers, we employed this method for the detection of CRP with a series of different concentrations in a sandwich immunoassay format. Because the particle-roughed surface of the PCB has a higher surface area than smooth ones, the dose-response range is from 500 fg mL-1 to 100 µg mL-1, covering more than 8 orders of magnitude (Figure 7a). And linear regression analysis result shows the biosensor has a linear response from 5 pg mL-1 up to 10 µg mL-1 with an R2 value of 0.96 (Figure 7b, inset), spanning 6 orders of magnitudes, which is wider than those of reported in previous studies (Table S-1 in the Supporting Information).53-56 The concentrations of CRP are typically less 3 µg mL-1 in the plasma of healthy humans and decrease to ng mL-1 or even pg mL-1 under numerous diseases and disorders.3, 57 Apparently, the LDR of this method covers this range. Therefore, when using the method, no dilution of the sample for pre-test is needed for quantitative detection and the time and reagent cost will be greatly decreased. This will be extremely valuable for high throughput protein marker detection. Meanwhile, the wider LDR will benefit greatly the detection in point of care testing (POCT), where pre-test is not practical. We then calculated the LOD and LOQ to be 478 fg mL-1 and 1.63 pg mL-1, respectively, which is also lower than that of the previously reported. The results demonstrate again the high signal to noise ratio and amplified signal intensity resulting from the combination of core-shell SERS nanotag and PCBs as mentioned above. In addition, the result of selectivity evaluation indicates that the biosensor exhibits excellent selection performance owing to the undesirable Raman signals are not detected on PCBs except in the CRP sample (Figure S-5 in the Supporting Information). Furthermore, the coefficient of variation of the assay among different batches is as low as 5.2 %. All these results demonstrate that our biosensor could be used for ultrasensitive detection of CRP with wide linear dynamic range, high stability and reproducibility. To demonstrate the clinical diagnosis, we employed this biosensor for the detection of real human serum samples as compared with the clinical reference method (ELISA). The concentrations of the five clinical samples are 7.7 µg mL-1, 1.42 µg mL-1, 162.5 ng mL-1, 528 ng mL-1 and 70.2 pg mL-1 respectively determined a

ACS Paragon Plus Environment

Page 12 of 18

Page 13 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

commercialized ELISA kit. We measured the samples directly with as-proposed biosensor. As shown in Figure 7c, the results obtained by the biosensor show good agreement with those acquired by ELISA because the correlation coefficient between the two methods for serum samples detection is 99.82 %, which indicate that the biosensor holds great promise for the detection of CRP with acceptable accuracy in real clinical serum samples.

Figure 7. CRP analysis with SERS nanotags and PCBs. (a) Raman spectra of different concentrations of CRP (from 500 fg mL-1 to 100 µg mL-1). (b) Reference plot of Raman intensities of Raman shift at 595 cm-1 vs. logarithm of CRP concentrations. Error bar is calculated from five repeats. The inset is the linear part of the reference plot. (c) Correlation of detection results acquired from the proposed biosensor (solid squares) and reference method ELISA (hollow squares) for the CRP in real serum samples. Error bar is calculated from five repeats.

CONCLUSIONS In conclusion, we have developed a biosensor combining Au@Ag NPs-based SERS nanotags and PCBs for ultrasensitive detection of protein with wide linear dynamic range. The results indicated that our biosensor exhibited good analytical performance with LOD for mouse IgG was 672 fg mL-1 and the linear dynamic detection range was from 10 pg mL-1 to 10 µg mL-1. In addition, for CRP detection, the LOD and LDR were 478 fg mL-1 and from 5 pg mL-1 to 10 µg mL-1, respectively, which is also much better than the reported. Moreover, the biosensor exhibited extremely low background, as well as high stability, reproducibility and reliability in real serum sample analysis. All these characteristics and advantages come from the combination of high surface to volume ratio of PCBs and high signal amplification of core-shell SERS nanotags, which also meets the requirements of analytical techniques for precision medical and provides potential applications in limited resource settings for the development of portable diagnostic.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Preparation of silica PCBs, determination of effective dielectric constant of the interior gap region, FDTD simulation models, and the supplemental figures and table (PDF)

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (No. 2017YFA0205700), National Natural Science Foundation of China (Grants 21373046, 21073033 and 21327902), Program Sponsored for Scientific Innovation Research of College Graduate in Jiangsu Province (KYLX16_0284), Jiangsu Science and Technology Department (Grant No. BE2014707), the Program for New Century Excellent Talents in University, Fundamental Research Funds for the Central Universities and Six Talent Peaks Project of Jiangsu Province. We are also very thankful to Shiya Zheng of Zhongda Hospital affiliated to Southeast University for providing real human serum samples.

REFERENCES (1) Whilde, J.; Martindale, M. Q.; Duffy, D. J. Precision wildlife medicine: applications of the human-centred precision medicine revolution to species conservation. Global Change Biol. 2017, 23, 1792-1805. (2) Rifai, N.; Gillette, M. A.; Carr, S. A. Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat. Biotech. 2006, 24, 971-983. (3) Vashist, S. K.; Venkatesh, A.; Schneider, E. M.; Beaudoin, C.; Luppa, P. B.; Luong, J. H. Bioanalytical advances in assays for C-reactive protein. Biotechnol. Adv. 2016, 34, 272-290. (4) Rusling, J. F.; Kumar, C. V.; Gutkind, J. S.; Patel, V. Measurement of biomarker proteins for point-of-care early detection and monitoring of cancer. Analyst 2010, 135, 2496-2511. (5) Tarkkinen, P.; Palenius, T.; Lövgren, T. Ultrarapid, ultrasensitive one-step kinetic immunoassay for C-reactive protein (CRP) in whole blood samples: measurement of the entire CRP concentration range with a single sample dilution. Clin. Chem. 2002, 48, 269-277. (6) Zhang, F.; Zhu, J.; Li, J. J.; Zhao, J. W. A promising direct visualization of an Au@Ag nanorod-based colorimetric sensor for trace detection of alpha-fetoprotein. J. Mater. Chem. C 2015, 3, 6035-6045. (7) Joung, H. A.; Oh, Y. K.; Kim, M. G. An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor. Biosens. Bioelectron. 2014, 53, 330-335. (8) Lee, J. S.; Joung, H. A.; Kim, M. G.; Park, C. B. Graphene-Based Chemiluminescence Resonance Energy Transfer for Homogeneous Immunoassay. Acs Nano 2012, 6, 2978-2983. (9) Gao, B.; Liu, H.; Gu, Z. Patterned Photonic Nitrocellulose for Pseudo-Paper Microfluidics. Anal. Chem. 2016, 88, 5424-5429. (10) Zeng, H.; Agyapong, D. A. Y.; Li, C.; Zhao, R.; Yang, H.; Wu, C.; Jiang, Y.; Liu, Y. A carcinoembryonic antigen optoelectronic immunosensor based on thiol-derivative-nanogold labeled anti-CEA antibody nanomaterial and gold modified ITO. Sens. Actuators, B 2015, 221, 22-27. (11) McRae, M. P.; Simmons, G.; Wong, J.; McDevitt, J. T. Programmable Bio-nanochip Platform: A

ACS Paragon Plus Environment

Page 14 of 18

Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

Point-of-Care Biosensor System with the Capacity To Learn. Accounts Chem. Res. 2016, 49, 1359-1368. (12) Fu, X.; Chen, L.; Choo, J. Optical Nanoprobes for Ultrasensitive Immunoassay. Anal. Chem. 2017, 89, 124-137. (13) Leng, Y.; Sun, K.; Chen, X.; Li, W. Suspension arrays based on nanoparticle-encoded microspheres for high-throughput multiplexed detection. Chem. Soc. Rev. 2015, 44, 5552-5595. (14) Andreou, C.; Kishore, S. A.; Kircher, M. F. Surface-Enhanced Raman Spectroscopy: A New Modality for Cancer Imaging. J. Nucl. Med. 2015, 56, 1295-1299. (15) Hakonen, A.; Andersson, P. O.; Schmidt, M. S.; Rindzevicius, T.; Kall, M. Explosive and chemical threat detection by surface-enhanced Raman scattering: A review. Anal. Chim. Acta 2015, 893, 1-13. (16) Hao, J.; Han, M. J.; Han, S.; Meng, X.; Su, T. L.; Wang, Q. K. SERS detection of arsenic in water: A review. J. Environ. Sci-China 2015, 36, 152-162. (17) Li, J.; Dong, S.; Tong, J.; Zhu, P.; Diao, G.; Yang, Z. 3D ordered silver nanoshells silica photonic crystal beads for multiplex encoded SERS bioassay. Chem. Commun. 2016, 52, 284-287. (18) Lim, D. K.; Jeon, K. S.; Hwang, J. H.; Kim, H.; Kwon, S.; Suh, Y. D.; Nam, J. M. Highly uniform and reproducible surface-enhanced Raman scattering from DNA-tailorable nanoparticles with 1-nm interior gap. Nat. Nanotechnol. 2011, 6, 452-460. (19) Liu, R.; Liu, B.; Guan, G.; Jiang, C.; Zhang, Z. Multilayered shell SERS nanotags with a highly uniform single-particle Raman readout for ultrasensitive immunoassays. Chem. Commun. 2012, 48, 9421-9423. (20) Samanta, A.; Jana, S.; Das, R. K.; Chang, Y. T. Biocompatible surface-enhanced Raman scattering nanotags for in vivo cancer detection. Nanomedicine 2014, 9, 523-535. (21) Rehm, H. L. Evolving health care through personal genomics. Nat. Rev. Genet. 2017, 18, 259-267. (22) Cao, Y. W.; Jin, R.; Mirkin, C. A. DNA-modified core-shell Ag/Au nanoparticles. J. Am. Chem. Soc. 2001, 123, 7961-7962. (23) Zhao, Y.; Yang, Y. X.; Luo, Y. D.; Yang, X.; Li, M. L.; Song, Q. J. Double Detection of Mycotoxins Based on SERS Labels Embedded Ag@Au Core-Shell Nanoparticles. Acs Appl. Mater. Inter. 2015, 7, 21780-21786. (24) Shen, J.; Su, J.; Yan, J.; Zhao, B.; Wang, D.; Wang, S.; Li, K.; Liu, M.; He, Y.; Mathur, S.; Fan, C.; Song, S. Bimetallic nano-mushrooms with DNA-mediated interior nanogaps for high-efficiency SERS signal amplification. Nano Res. 2015, 8, 731-742. (25) Wang, H.; Guo, X.; Fu, S.; Yang, T.; Wen, Y.; Yang, H. Optimized core-shell Au@Ag nanoparticles for label-free Raman determination of trace Rhodamine B with cancer risk in food product. Food Chem. 2015, 188, 137-142. (26) Tang, B.; Zhao, X.; Zhao, Y.; Zhang, W.; Wang, Q.; Kong, L.; Gu, Z. Binary optical encoding strategy for multiplex assay. Langmuir 2011, 27, 11722-11728. (27) Liu, B.; Zhao, X.; Jiang, W.; Fu, D.; Gu, Z. Multiplex bioassays encoded by photonic crystal beads and SERS nanotags. Nanoscale 2016, 8, 17465-17471. (28) Zhao, X.; Cao, Y.; Ito, F.; Chen, H. H.; Nagai, K.; Zhao, Y. H.; Gu, Z. Z. Colloidal crystal beads as supports for biomolecular screening. Angew. Chem. Int. Ed. 2006, 45, 6835-6838. (29) Zhao, X.; Zhao, Y.; Hu, J.; Xu, M.; Zhao, W.; Gu, Z. Sintering Photonic Beads for Multiplex Biosensing. J. Nanosci. Nanotechnol. 2010, 10, 588-594. (30) Yang, Z.; Xie, Z.; Liu, H.; Yan, F.; Ju, H. Streptavidin-Functionalized Three-Dimensional Ordered Nanoporous Silica Film for Highly Efficient Chemiluminescent Immunosensing. Adv. Funct. Mater. 2008, 18, 3991-3998.

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(31) Frens, G. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nat. Phys. Sci. 1973, 241, 20-22. (32) Xie, Y.; Chang, H.; Zhao, K.; Li, J.; Yang, H.; Mei, L.; Xu, S.; Deng, A. A novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering for the sensitive and quantitative determination of clenbuterol. Anal. Methods 2015, 7, 513-520. (33) Liu, B.; Han, G.; Zhang, Z.; Liu, R.; Jiang, C.; Wang, S.; Han, M. Y. Shell Thickness-Dependent Raman Enhancement for Rapid Identification and Detection of Pesticide Residues at Fruit Peels. Anal. Chem. 2012, 84, 255-261. (34) Lin, L.; Zapata, M.; Xiong, M.; Liu, Z. H.; Wang, S. S.; Xu, H.; Borisov, A. G.; Gu, H. C.; Nordlander, P.; Aizpurua, J.; Ye, J. Nanooptics of Plasmonic Nanomatryoshkas: Shrinking the Size of a Core-Shell Junction to Subnanometer. Nano Lett. 2015, 15, 6419-6428. (35) Tan, T.; Tian, C.; Ren, Z.; Yang, J.; Chen, Y.; Sun, L.; Li, Z.; Wu, A.; Yin, J.; Fu, H. LSPR-dependent SERS performance of silver nanoplates with highly stable and broad tunable LSPRs prepared through an improved seed-mediated strategy. Phys. Chem. Chem. Phys. 2013, 15, 21034-21042. (36) Hu, Y.; Noelck, S. J.; Drezek, R. A. Symmetry Breaking in Gold−Silica−Gold MulJlayer Nanoshells. Acs Nano 2010, 4, 1521-1528. (37) Zhu, J.; Ren, Y. J.; Zhao, S. M.; Zhao, J. W. The effect of inserted gold nanosphere on the local field enhancement of gold nanoshell. Mater. Chem. Phys. 2012, 133, 1060-1065. (38) Krug, J. T.; Wang, G. D.; Emory, S. R.; Nie, S. Efficient Raman Enhancement and Intermittent Light Emission Observed in Single Gold Nanocrystals. J. Am. Chem. Soc. 1999, 121, 9208-9214. (39) Neng, J.; Harpster, M. H.; Wilson, W. C.; Johnson, P. A. Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles. Biosens. Bioelectron. 2013, 41, 316-321. (40) Olson, T. Y.; Schwartzberg, A. M.; Orme, C. A.; Talley, C. E.; O'Connell, B.; Zhang, J. Z. Hollow gold-silver double-shell nanospheres: Structure, optical absorption, and surface-enhanced Raman scattering. J. Phys. Chem. C 2008, 112, 6319-6329. (41) Bruzzone, S.; Arrighini, G. P.; Guidotti, C. Electromagnetic response behavior of binary metallic nanoparticles: a comparison of results from a few models of different schematicity. Mater. Sci. Eng. C 2003, 23, 191-200. (42) Shi, H. Z.; Zhang, L. D.; Cai, W. P. Composition modulation of optical absorption in AgxAu1-x alloy nanocrystals in situ formed within pores of mesoporous silica. J. Appl. Phys. 2000, 87, 1572-1574. (43) Lee, S.; Kim, S.; Choo, J.; Shin, S. Y.; Lee, Y. H.; Choi, H. Y.; Ha, S.; Kang, K.; Oh, C. H. Biological imaging of HEK293 cells expressing PLC gamma 1 using surface-enhanced raman microscopy. Anal. Chem. 2007, 79, 916-922. (44) Lassiter, J. B.; Aizpurua, J.; Hernandez, L. I.; Brandl, D. W.; Romero, I.; Lal, S.; Hafner, J. H.; Nordlander, P.; Halas, N. J. Close Encounters between Two Nanoshells. Nano Lett. 2008, 8, 1212-1218. (45) Prodan, E.; Radloff, C.; Halas, N. J.; Nordlander, P. A Hybridization Model for the Plasmon Response of Complex Nanostructures. Science 2003, 302, 419-422. (46) Lin, L.; Gu, H.; Ye, J. Plasmonic multi-shell nanomatryoshka particles as highly tunable SERS tags with built-in reporters. Chem. Commun. 2015, 51, 17740-17743. (47) Chen, G. Y.; Roy, I.; Yang, C. H.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826-2885. (48) Zhu, X.; Li, J.; He, H.; Huang, M.; Zhang, X.; Wang, S. Application of nanomaterials in the bioanalytical detection of disease-related genes. Biosens. Bioelectron. 2015, 74, 113-133.

ACS Paragon Plus Environment

Page 16 of 18

Page 17 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

(49) He, S.; Li, X.; Gao, J.; Tong, P.; Chen, H. Development of sandwich ELISA for testing bovine β-lactoglobulin allergenic residues by specific polyclonal antibody against human IgE binding epitopes. Food Chem. 2017, 227, 33-40. (50) Chen, Z.; Liang, R.; Guo, X.; Liang, J.; Deng, Q.; Li, M.; An, T.; Liu, T.; Wu, Y. Simultaneous quantitation of cytokeratin-19 fragment and carcinoembryonic antigen in human serum via quantum dot-doped nanoparticles. Biosens. Bioelectron. 2017, 91, 60-65. (51) Mu, Z.; Zhao, X.; Huang, Y.; Lu, M.; Gu, Z. Photonic Crystal Hydrogel Enhanced Plasmonic Staining for Multiplexed Protein Analysis. Small 2015, 11, 6036-6043. (52) Zhao, X.; Xue, J.; Mu, Z.; Huang, Y.; Lu, M.; Gu, Z. Gold nanoparticle incorporated inverse opal photonic crystal capillaries for optofluidic surface enhanced Raman spectroscopy. Biosens. Bioelectron. 2015, 72, 268-274. (53) Joung, H. A.; Oh, Y. K.; Kim, M. G. An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor. Biosens. Bioelectron. 2014, 53, 330-335. (54) Yang, S.; Gao, B.; Tsai, H.; Fuh, C. B. Detection of c-reactive protein based on a magnetic immunoassay by using functional magnetic and fluorescent nanoparticles in microplates. Analyst 2014, 139, 5576-5581. (55) Bryan, T.; Luo, X.; Bueno, P. R.; Davis, J. J. An optimised electrochemical biosensor for the label-free detection of C-reactive protein in blood. Biosens. Bioelectron. 2013, 39, 94-98. (56) Zhang, X.; Hu, R.; Zhang, K.; Bai, R.; Li, D.; Yang, Y. An ultrasensitive label-free immunoassay for C-reactive protein detection in human serum based on electron transfer. Anal. Methods 2016, 8, 6202-6207. (57) Byun, J. Y.; Shin, Y. B.; Kim, D. M.; Kim, M. G. A colorimetric homogeneous immunoassay system for the C-reactive protein. Analyst 2013, 138, 1538-1543.

ACS Paragon Plus Environment

ACS Sensors

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For TOC only

ACS Paragon Plus Environment

Page 18 of 18