Specific Detection of Carcinoembryonic Antigen Based on

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX-XXX

Specific Detection of Carcinoembryonic Antigen Based on Fluorescence Quenching of Hollow Porous Gold Nanoshells with Roughened Surface Ting-Yang Xing, Jing Zhao, Guo-Jun Weng, Jian Zhu, Jian-Jun Li,* and Jun-Wu Zhao* The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China ABSTRACT: The detection of tumor biomarkers in the early stage is highly desirable for the therapy of cancer. However, rapid, low-cost, sensitive, and selective detection of biomarkers remains a challenge owing to the sequence homology, short length, and low abundance. This Research Article describes the synthesis of a novel carcinoembryonic antigen (CEA) probe using hollow porous gold nanoparticles (HPGNPs) with roughened surface based on fluorescence quenching. For specific detection of CEA, the surface of HPGNP is modified by carboxyl modification, carboxyl activation, and antibody conjugation. Furthermore, to enhance the detection performance, we have systematically optimized the parameters, such as particle size, surfactants, surface roughness, surface hole size, and the molecule-particle distance (MPD). The results demonstrate that the fluorescence quenching efficiency would be enhanced with a larger particle size and surface hole size, roughened surface and a greater MPD. Also, with careful inspection of different surfactants of CTAB and PVP, we find that PVP has the optimal performance on fluorescence quenching. Under these optimized conditions, CEA could be detected with an ultralow detection limit of 1.5 pg/mL, and the probe shows a linear range from 2 to 100 pg/mL. The limit of detection is an order of intensity lower than related methods. Interference experiment results have shown that the influence of the interfering proteins could be neglected in the detection procedure. KEYWORDS: hollow porous gold nanoparticles, surface roughness, specific detection, fluorescence quenching, carcinoembryonic antigen

1. INTRODUCTION

Due to the unique optical properties of nanoparticles, nanotechnology has played an increasingly significant role in diverse fields ranging from the catalyst,14 advanced material synthesis,15 to preventative medicine.16 In the area of medical biosensors, considerable efforts have been made, and signal transducers such as fluorescence,15,17−19 biobarcodes,20 Raman dyes,21 and enzymes22 for signal amplification have been created. Among these methods, the development of biosensors based on fluorescence, especially fluorescence quenching has given rise to considerable attention owing to the ultrahigh sensitivity and the lower background signals compared to traditional molecular approaches.23,24 The metal induced fluorescence quenching (MIFQ) is based on the nonradiative energy transformation, as a result of the surface plasmon resonance (SPR) induced fluorescence quenching when near to metallic nanoparticles. The distance between the fluorescence molecules and the metal nanoparticles determines the occurrence of either fluorescence quenching or fluorescence enhancement. In comparison to fluorescence enhancement, which has very stringent requirement on the distance,25,26 fluorescence quenching is easier to be observed and has been

Cancer is the second leading cause of death globally, in 2015, the World Health Organization (WHO) estimate cancer is accounted for 8.8 million deaths. They also indicate that the early diagnosis of cancer is important to improve the likelihood of successful treatment. Thus, the development of selective, accurate and sensitive biosensors for the detection of cancer markers is of increasing importance.1,2 In the last few decades, many detection strategies become available, such as ELISAbased methods, electrochemical and electrical detection methods, optical methods and some others methods.3−6 However, most existing methods or biosensors suffer from some drawbacks, including the need for a long experimental period, poor selectivity or a high detection limit.7,8 Among these methods, enzyme-linked immunosorbent assay (ELISA) is the gold standard in the detection of various target biomarkers, especially proteins, based on the specific and high recognition of antibodies to their corresponding antigens.9,10 However, ELISA is time-consuming and the operations require a well-trained technician and special equipment in a laboratory.11 Besides, when the concentration of biomarkers in biological samples from patients is less than nanomolar,12,13 ELISA will not be efficient. Therefore, it is urgently required to fabricate biosensors with ultrasensitivity in the detection of biomarkers. © XXXX American Chemical Society

Received: July 31, 2017 Accepted: October 4, 2017

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DOI: 10.1021/acsami.7b11310 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Scheme 1. Surface Modification Procedures of Hollow Porous Gold Nanoparticles Including Carboxyl Replacement, Carboxyl Activation, and Antibody Connection

based on HPGNPs. At present, studies of the effect on fluorescence quenching efficiency of nanoparticles mainly focus on the morphology and size. To improve the fluorescence quenching efficiency and sensitivity, we have systematically investigated various kinds of nanoparticle parameters such as particle size, surfactants, surface hole size, surface roughness, and the distance between molecules and gold nanoparticles. Our experimental results showed that HPGNPs could exhibit enhanced fluorescence quenching efficiency with larger particle size, surface hole size, roughened surface and the moleculeparticle distance. Besides, in comparison with different surfactants, PVP is preferred in the preparation of the fluorescence probe. For the specific detection of CEA, we have modified the surface of HPGPNs with the antibody. Based on our findings, CEA could be detected with an ultralow detection limit of 1.5 pg/mL with a linear range from 2 to 100 pg/mL.

extensively studied and employed as in the fabrication of medical sensors. When the distance remains constant, the fluorescence emission properties of fluorescent molecules in MIFQ are expected to depend critically on many factors such as the size and shape of nanoparticle, the acceptor surface roughness, the orientation of molecular dipole with respect to the moleculeparticle axis, and the overlap of molecular emission with particle’s absorption spectrum. Dulkeith et al. demonstrated that the fluorescence quenching yield increases with the increase of gold nanoparticles radius,26,27 The efficiency of energy transfer is also shape-dependent, spherical gold nanoparticles were shown to have more efficient energy transfer according to the quenching constants in comparison to gold nanorod, gold nanotriangle, and gold nanobipyramids. To investigate the influence of surface roughness on the radiative and nonradiative energy transfer, metal island film28,29 or electrode with rough surfaces30 have been prepared, and it was evident that the rough surface could enhance the fluorescence quenching due to the surface-damping effect below a critical intermolecular distance. Compared to another kind of metal nanoparticles, hollow porous nanoshells allow the penetration of fluorescent molecules into the hollow interior, leading to the enhancement of fluorescence quenching efficiency.31 The most commonly used mechanism for synthesizing hollow metal nanoparticles is the combination of Ostwald Ripening and redox reaction, using Ag nanoparticle as the sacrificial template. The choice of surfactants is important for both the redox reaction and the subsequent fluorescence quenching. Jinchuan et al. have shown that citrate could enhance the fluorescence intensity of carboxyfluorescein in colloid gold solution and correspondingly would reduce the fluorescence quenching efficiency.32 The removal of citrate led to a 10-fold increase in the fluorescence quenching efficiency; however, the gold nanoparticles are prone to aggregation without surfactants. Xia’s group uses PVP as the surfactants for the preparation of Ag template and the following reaction of synthesizing hollow gold nanocubes.33 In our previous report, sodium citrate is used in the preparing of Ag nanoparticles, and CTAB is used in the fabrication of hollow gold nanoparticles.34 Both CTAB and PVP are commonly used surfactants in preparing hollow gold nanoparticles. However, the influence of PVP and CTAB on the fluorescence quenching efficiency remains uninvestigated. Here, we prepare hollow porous gold nanoparticles by selectively etching hollow gold nanoparticles (HGNPs) at critical state using Fe(NO3)3 and find that HPGNPs have a high quenching efficiency of carcinoembryonic antigen fluorescence. Thus, a novel CEA probe have been synthesized

2. MATERIAL AND METHODS 2.1. Materials and Reagents. Polyvinylpyrrolidone K30 (PVP K30) was purchased from Tokyo Chemical Industry Co., Ltd. (TCI). Iron(III) nitrate nonahydrate (Fe(NO3)3·9H2O) and chloroauric acid tetrahydrate (HAuCl4·4H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd., China. Trisodium citrate (Na3C6H5O7), tannic acid (C76H52O46), silver nitrate(AgNO3, >99%, Sigma-Aldrich), mercaptoacetic acid (TGA, 98%, C2H4O2S) produced by Acros and 16-mercaptohexadecanoic acid (16-MHDA, HS(CH2)15CO2H) produced by Sigma was purchased from Aladdin Industrial Corporation. N-Hydroxysuccinimide (NHS) was acquired from J&K Scientific, Ltd. 1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) was purchased from BIO BASIC Inc. Rabbit antirat serum albumin (IgG) and α-L-fucosidase (AFU) were obtained from Beijing Biosynthesis Biotechnology Co., Ltd. Bovine serum albumin (BSA) was purchased from Xi‘an voerson biological technology Co., Ltd., Alpha-fetoprotein (AFP), CEA test kits was acquired from Zhengzhou Biocell Biotechnology Co., Ltd. and anti-CEA test kits were acquired from Bioss. Deionized water (Millipore Milli-Q grade) with a resistivity of 18.2 MΩ cm was used throughout this study. 2.2. Synthesis of Hollow Porous Gold Nanoparticles. First, with the combination of sodium citrate and tannic acid, silver nitrate was reduced, and Ag nanospheres (AgNSs) with average diameters of 40, 47, 51 nm were prepared.35 Then, the AgNSs were centrifuged at 10000 rpm for 10 min. To fabricate PVP and CTAB coated AgNSs, the AgNSs were resuspended into corresponding surfactants. It should be noted that the concentrations of PVP and CTAB solutions are 0.1 g/mL and 0.1 M. After that, silver nanospheres were heated with intense stirring. When the temperature reaches 80 °C, chloroauric acid tetrahydrate was added dropwise.33 The absorption spectra of nanoparticles colloid were collected every 2 min, and the heating was halted when the first absorption spectrum peak of Au−Ag alloy was disappeared which indicating the interior block Ag of hollow Au− Ag nanoparticles reduced entirely by Au.36 Finally, the above-obtained B


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ACS Applied Materials & Interfaces hollow Au−Ag nanoparticles were etched by the selective etching of Ag using Fe(NO3)3 and hollow porous gold nanoparticles were prepared.37 In this study, the surface hole size of HPGNPs could be tuned with different amount of Fe(NO3)3. 2.3. Surface Modification of Hollow Porous Gold Nanoparticles. For the elimination of redundant Fe(NO3)3, HPGNPs were cleaned by centrifugation at 10000 rpm for 10 min and every 3 mL Au colloid was added into different sample bottles. Then, surface modification was carried out involving three steps as shown in Scheme 1. (1) Carboxyl replacement: 100 μL TGA or 16-MHDA ethanol solution of 20 mM was injected into the sample bottles as required and the resultant solution was treated with ultrasonic oscillation (35 °C for 0.5 h and 45−50 °C for 3 h). Afterward, to remove the unreacted TAG or 16-MHDA, the mixture was centrifuged at 7000 rpm for 10 min and resuspended in 3 mL PVP solution (10 mg/mL). (2) Carboxyl activation: 100 μL EDC of 80 mM and 100 μL NHS are added into the samples and kept at room temperature for 20 min. (3) Antibody connection: anti-CEA was added in each resultant solution with a final concentration of 4 μg/mL and antibody coupling could be finished in hours.38 2.4. Detection of CEA. On the basis of the above-described protocol, hollow porous gold nanoparticles were modified and used for the detection of CEA. After the addition interfering proteins, to remove excessive PVP and unbound proteins, the well-prepared CEA probe should be cleaned by centrifugation at 5000 rpm for 4 min and resuspended in ultrapure water. To preserve the dispersibility of the solution, the centrifugal speed and duration time should be precisely controlled. Because the Raman signals of water are stronger than that of CEA when the CEA concentration is lower than 2 pg/mL, and which would substantially decrease the readability of signals. Thus, fluorescence spectra were recorded from 320−500 nm. 2.5. Characterization. UV−vis absorption spectra were obtained using a Shimadzu UV-3600 UV/vis/near-IR spectrometer. Fluorescence emission spectra were acquired from a FluoroMax-4P spectrophotometer (HORIBA Jobin Yvon Inc., France) with the excitation at 280 nm. HRTEM images and energy dispersive X-ray spectroscopy (EDS) were obtained by an JEM-2100Plus transmission electron microscope (JEOL, Japan). Centrifugation was carried out by using a 5810 Rcentrifuge (Eppendorf, Japan). A Milli-Q water purification system (Millipore, USA) was used to acquire the deionized water.

the combination of sodium citrate and tannic acid. Because sodium citrate and tannic acid would compete with Ag(s) in the following reduction reaction, they were removed from the silver colloid solution by centrifugation. To ensure the stability of the reaction system in next steps (i.e., the reduction reaction), silver nanospheres were resuspended in ultrapure water containing different surfactants such as CTAB and PVP. The hollow gold nanoparticles were prepared by the redox process based on the fact that the standard potential of AuCl4−/Au pair (0.99 V, vs SHE) is higher than that of Ag+/Ag pair (0.80 V, vs SHE). In our experiments, the redox reaction is carried out by heating the solution to 80 °C followed by dropwise addition of 1 mM HAuCl4 under vigorous stirring. The disappearance of the plasmon resonance peak of Ag nanoparticles indicates a critical state of Au−Ag alloy nanoparticles, and the continuous addition of HAuCl4 will lead to the formation of hollow gold nanoparticles with seamless shells. However, as the hollow porous gold nanoparticles are needed, hence, we should stop the addition of HAuCl4 at the critical state and selectively etch Ag of Au−Ag alloy nanoparticles with Fe(NO3)3. 3.2. Effect of Particle Size on Fluorescence Quenching Efficiency. To observe the optical properties of HPGNPs, PVP-coated hollow porous gold nanoparticles of different sizes have been prepared from silver templates and the absorption spectra of which are shown in Figure 1A. It can be observed from the spectra that the major absorption peaks which are corresponding to the SPR of HPGNPs locate within the nearinfrared region. As the diameters of the silver templates increase from 45 to 56 nm, a redshift of the major absorption peaks from 785 to 832 nm can be observed. Then, we have evaluated the fluorescence quenching efficiency the HPGNPs using CEA. In our experiments, the fluorescence spectra of CEA were excited by laser with a wavelength of 280 nm. As shown in Figure 1B, the fluorescence spectra demonstrate that HPGNPs of larger size exhibit higher fluorescence quenching efficiency. For this spherical nanoparticles, the experimental results presented good consistency with the Silber and Kuhn (CPSKuhn) model which is currently the most valuable model in understanding how different parameters affect the behavior of an emitting dipole in proximity to metal nanomaterials.39 3.3. Effect of Surfactants and Surface Roughness on Fluorescence Quenching Efficiency. During the preparing process of hollow porous gold nanoparticles, the non-HPGNP component which consists mainly the free surfactants, such as CTAB and PVP cannot be completely removed, hence, their effect on the fluorescence quenching efficiency worth to be evaluated. The non-HPGNPs component on the sensitivity of CEA detection were systematically studied by evaluating the fluorescence quenching efficiency of the supernatant that was collected after the reaction mixture was centrifuged at 10000 rpm for 10 min. As shown in Figure 2, the influence of supernatants from HPGNPs coated with different surfactants were investigated using CEA at two concentrations (1 pg/mL and 12 pg/mL). From Figure 2A, we can see that, when the CEA level is 1 pg/mL, supernatants containing CTAB would increase the fluorescence intensity by up to 3 times, in contrary, the supernatants containing PVP reduces the fluorescence intensity. Besides, compared to the fluorescence spectrum of CEA in ultrapure water, fluorescence spectra are blue-shifted by ∼10 nm for supernatants containing CTAB and red-shifted by ∼50 nm for that containing PVP. When the CEA concentration was increased to 12 pg/mL as shown in Figure 2B, fluorescence intensity would increase by up to ∼1.5 times for supernatants

3. RESULTS AND DISCUSSION 3.1. Fabrication of Hollow Porous Gold Nanoparticles. In this study, the preparation of the carcinoembryonic antigen probe based on fluorescence quenching is composed of two parts: the preparation of hollow porous gold nanoparticles and the parameters optimization. First, hollow gold nanoparticles can be prepared using the protocol as shown in Scheme 2. The Ag nanospheres were prepared by reducing silver nitrate using Scheme 2. Schematic Illustration of the Preparation Procedures of Hollow Gold Nanoparticles and Hollow Porous Gold Nanoparticles (HPGNP)

C


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Figure 1. (A) Normalized absorption of hollow porous gold nanoparticles of different sizes and (B) fluorescence emission spectra of CEA quenched by water and hollow porous gold nanoparticles of different sizes.

Figure 2. Effect of different surfactants on fluorescence emission spectra of CEA with concentrations of (A) 1 and (B) 12 pg/mL.

Figure 3. (A) Absorption spectra of hollow gold nanoparticles resuspended in CTAB and PVP and (B) fluorescence emission spectra of CEA quenched by hollow porous gold nanoparticles with roughened surface coated by corresponding surfactants.

containing CTAB and decrease by ∼81% for that containing PVP. Concerning fluorescence peak position, in supernatant containing CTAB, the peak position still blue-shifted by ∼10 nm, however, in supernatant containing PVP, the peak position red-shifted to a lesser extend which is around ∼15 nm. Regardless of the exact number, the general trend remains unchanged in different concentrations. Therefore, we can conclude that CTAB has an enhancement effect and PVP has a weakening effect on the fluorescence of CEA in spite of the concentration. In our application which uses fluorescence quenching for the detection of CEA, PVP tends to be a more appropriate coating material to improve the detection sensitivity. The possible reasons that explain why CTAB and PVP have different effects on the fluorescence spectrum of CEA are

shown as follows. In CEA, there are three amino acid residues that are primarily responsible for the inherent fluorescence of proteins, and they are tryptophan, tyrosine, and phenylalanine. Tryptophan is much more fluorescent than either tyrosine or phenylalanine. However, the fluorescent properties of tryptophan are solvent dependent. Surfactants like CTAB are expected to decrease the polarity of the solution. The decrease in polarity of solvent results in a blue-shift of fluorescence spectra and an increase in spectra intensity. This is because in polar solvents, tryptophan tends to be buried in the hydrophobic domains of folded proteins, and therefore exhibit a spectral shift of 10−20 nm. Therefore, a blue-shift of ∼10 nm and an enhanced intensity of CEA fluorescence spectra in supernatants containing CTAB is observed. Concerning PVP, the hydrophilic alkyl chain can bind a large number of water D


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Figure 4. (A) Hollow porous gold nanoparticles with the smooth surface using CTAB as a surfactant and (B) rough surface using PVP as a surfactant.

Figure 5. (A) Absorption spectra of hollow porous gold nanoparticles with different amount of Fe(NO3)3 and the particle size is 56 nm. (B) Fluorescence emission spectra of CEA with concentration 20 pg/mL in HPGNPs solution containing different amount of Fe(NO3)3.

coated by CTAB is larger than that coated by PVP, or in another word, PVP-coated HPGNPs have greater fluorescence quenching efficiency. Thus, to get a larger fluorescence quenching efficiency in the preparation of biomarker sensors, the use of PVP as the surfactants is recommended. To investigate whether the coating of surfactants indeed affects the surface morphology of HPGNPs, TEM images of HPGNPs coated by CTAB and PVP have been taken as shown in Figure 4. It can be observed that the surface of HPGPNs coated with CTAB is more smooth and the wall thickness is larger than HPGPNs coated with PVP. Besides, with careful inspection, HPGNPs coated with PVP have more steps on their surface. Because the coarser surface could increase the uniformity of HPGNPs and slightly change the SPR modes, the increased roughness is the main reason that led to the broadening of the absorption spectrum of PVP-coated HPGNPs. The greater wall thickness of CTAB coated HPGNPs is responsible for the shorter wavelength of absorption peaks position, due to that the absorption peak position of HPGNPs with the same size but a thinner wall thickness is longer. On the other hand, as shown in Figure 4A, more HPGNPs using CTAB as surfactants have cracked compared to that using PVP because of the increasing etching rate induced by the Br− in solution.43 3.4. Effect of Surface Hole Size and Quantities on Fluorescence Quenching Efficiency. Because the surface hole size of HPGNPs is the primary factor controlling the biomarkers transportation, which plays a significant role in the fluorescence quenching efficiency, hence, HPGNPs with different surface hole sizes have been prepared. In our

molecules to quench the excited states of CEA through the vibrational energy of the OH groups.40 The quenching of protein fluorescence has also been reported by Jiang et al. that when the concentration of PVP exceeded 0.6%, the fluorescence of bovine carbonic anhydrase B (CAB) is decreased with a prominent spectral red-shift.41 Therefore, we postulate the red-shift of CEA fluorescence spectra is due to the high PVP concentration which leading to the denaturation CEA. On the basis of the above description, HPGNPs coated by PVP are expected to have a higher quenching efficiency compared to that coated by CTAB. To verify this postulation, HPGNPs coated with CTAB and PVP were prepared. Different from previous processes, HPGNPs were centrifuged, and the supernatants were carefully aspirated to minimize the effect of free surfactants on the fluorescence quenching efficiency. From Figure 3A, we can see that in comparison with absorption peaks of CTAB coated HPGNPs, which coated by PVP have a redshift of ∼90 nm. Besides, the absorption spectra of PVPcoated HPGNPs have a larger full width at half-maximum. This red-shifted and border absorption spectra may be ascribed to a number of factors. First, the different molecular structures and mass fractions have different effects on the refractive index near the surface of HPGNPs. The mass fractions of PVP are larger than that of CTAB, this is accounted for the greater refractive index and lead to the longer absorption peak. On the other hand, surfactants around the surface of Ag nanoparticles have influences on the formation of surface morphology of HPGNPs.42 As shown in Figure 3B, the results demonstrated that fluorescence intensity of CEA (20 pg/mL) in HPGNPs E


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Figure 6. TEM images of hollow porous gold nanoparticles with different addition of Fe(NO3)3 (A) 0, (B) 1, (C) 2, and (D) 3 μmol.

Figure 7. EDS elemental mapping of HPGNPs etched using Fe3+ of (A) 0, (B) 1, (C) 2, and (D) 3 μmol. (E) Ratio of Ag and Au atomic as a function of the quantities of Fe3+.

fluorescence quenching efficiency was enhanced when the amount of Fe(NO3)3 was increased. The phenomenon could be attributed to the reason that larger surface hole size can enhance the permeation of molecules, and increase the surface area and fluorescence quenching efficiency. Furthermore, to directly observe the surface structure of PVP-coated hollow porous gold nanoparticles with different amounts of Fe(NO3)3 added in the solution, high-resolution TEM images were taken as shown in Figure 6. In Figure 6A, we could see that when no Fe(NO3)3 was added, hollow gold nanoparticles were at the critical state and no surface holes could be observed. The dark interior in hollow porous gold nanoparticles denotes the Ag(s), therefore, the gradual brightening of the interior from Figure 6A−6D indicate the gradual reduction of Ag(s). As shown in Figure 6C and 6D, the small and white spots on the surface are holes. Therefore, it can

experiments, the surface hole size was tuned by varying the amounts of etchant Fe(NO3)3. It can be observed from Figure 5A that the absorption peak of hollow gold nanoparticles at the critical state is 772 nm without adding Fe(NO3)3. Addition of Fe(NO3)3 lead to the selective etching of Ag(s), and an increase in the size of the surface holes, which then resulted in the redshifts of the absorption peak. However, when the amount of Fe(NO3)3 added is larger than 0.3 μmol, no further redshift of the absorption peak could be observed. It is possibly due to the reason that Ag(s) both on the surface and in the interior of the hollow porous gold nanoparticles have been completely eliminated, and the addition of excessive amount of Fe(NO3)3 had no effect on the surface morphology, and therefore the position of the absorption peak. By using CEA of 20 pg/mL, fluorescence quenching efficiency of HPGNPs was evaluated as shown in Figure 5B. The results revealed that the F


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Figure 8. (A) Absorption spectra of hollow porous gold nanoparticles at different surface modification steps. (B) Fluorescence emission spectra of CEA of 20 pg/mL with various distances tuned by TGA and 16-MHDA.

Figure 9. Fluorescence quenching efficiency of HPGNPs as a function of CEA concentration. (A) CEA concentration has a large region between 0.5 and 140 pg/mL. (B) CEA concentration has a linear region between 2 and 100 pg/mL.

mercaptan acid with carbon chains of different lengths. Here, TGA and 16-MHDA were used to evaluate the effect of distance on fluorescence quenching efficiency. From Figure 8A, we can see the absorption spectra of HPGNPs of different modifying stages. When HPGNPs were modified with TGA, the absorption spectrum red-shifted by ∼25 nm, which is attributed to the increasing refractive index around the surface of HPGNPs as the Au−S bond is formed during the modification. The antibody was conjugated to the TGAmodified HPGNP by carbodiimide chemistry using EDC and NHS as catalyst. The carboxyl group of TGA was first activated to a reactive o-acylisourea ester by EDC and then to more stable amine-active NHS ester by NHS. Then, antibodies of CEA were added into the mixtures, and the absorption spectra of the resultant solution showed a redshift ∼15 nm which indicated that CEA had been connected. For comparison, CEA was conjugated to 16-MHDA modified HPGNPs using the same protocol except that the TGA was replaced by 16-MHDA with the chain length increased to C16. It can be observed from the fluorescence spectra in Figure 8B that the quenching efficiency of HPGNPs modified using TGA is larger than that modified using 16-MHDA. Because the carbon length equals to the result of carbon numbers multiplied by ∼0.125 nm, therefore, the molecule-particle distance can be adjusted from C + 0.25 nm to C + 4 nm, where C means the length of other materials. For molecules in the vicinity of the gold nanoparticles, fluorescence excitation rate is increased with an enhanced local field. However, the nonradiative energy transfer to the particles leads to a decrease in the quantum yield (quenching). In this competitive relation, the molecule-particle

be observed from the images that when the amount of Fe(NO3)3 was increased from 1 to 3 μmol, the quantity and the size of surface holes become large. This is ascribed to the etching of Ag(s) on the surface of Au−Ag alloy. Besides, it could also be seen from the images that the surface holes are randomly and uniformly distributed with increasing quantity and size when the Fe(NO3)3 were gradually increased. To study the components of hollow gold nanoparticles with the increasing addition of Fe3+, EDS elemental mapping images have been token as shown in Figure 7. The clear color contrast of Ag (green) and Au (red) implies that the surface of HPGNPs is alloyed. Besides, we can see that points of green color become more sparse in the first row from Figure 7A to 7D which indicates the decreasing content of Ag(s) in the particles. In contrast, the change of red color points is not significant. Figure 7E plots the ratio of Ag and Au atomic as a function of the amounts of Fe3+. The figure shows that the ratio of Ag and Au atomic decreases from 4.25 to 1.65 when the amounts of Fe3+ increase from 0 to 3 μmol and change little when the amounts of Fe3+ are exceed 3 μmol. In Figure 5A, we have observed that there is no further redshift of the absorption peak when the amounts of Fe3+ are larger than 3 μmol. Therefore, we infer that the remanent Ag(s) plays the role of supporting the framework of the nanoparticles and further etching will lead to the crack the nanoparticles. 3.5. Effect of Molecule−Particle Distance on the Fluorescence Quenching Efficiency. Using the surface modification procedures as shown in Scheme 2 and the optimized nanoparticles parameters, the distance between gold nanoparticles and CEA antigen molecules were tuned using G


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selectively detected CEA in the range of 0.1−60 ng/mL with a detection limit of 0.03 ng/mL. Although Zhou’s work has the highest upper detection limit, the lower detection limit is much higher than that in our work, hence, this method may not be able to detect the trace amount of CEA in the early stage of diseases. Using a gold-patterned microarray chip based on surface-enhanced Raman scattering imaging, Chen et al. prepared an immunoassay of cancer markers which had a wider linear range from 5 pg/mL to 500 μg/mL and a detection limit of 5 pg/mL. As we can know, the detection limit they reported is the lowest in literature and is comparable to ours. However, their method is time-consuming, and the preparation of microarray has much higher complexity and cost than our method. Therefore, the fast, simple and cost-effective fluorescence quenching method in this work extends the lower detection limit to 1.5 pg/mL, which is lower than that in current literature, and holds great promise in the early detection of CEA. Specificity, which is the resistance to interference from irrelevant contaminants, is one of the most important parameters for the evaluation of a sensor. To test the specificity of this probe, the fluorescence quenching efficiencies of CEA, BSA, AFP, IgG, and AFU by hollow porous gold nanoparticles have been evaluated. In the interfering experiment, the concentrations of CEA are 2 pg/mL and all interfering proteins are 10 μg/mL. As shown in Figure 10A, the fluorescence of CEA could be mostly quenched, and the quenching efficiency reaches up to 0.93. This is because CEA antigen can be selectively attached to the CEA antibody modified hollow porous gold nanoparticles, and then the fluorescence is quenched. In contrast, molecules like BSA, AFP, IgG, and AFU can only interact with the nanoparticles via nonspecific binding. Therefore, the quenching efficiency is relatively low. In Figure 10B, the interference of other proteins on the fluorescence quenching efficiency of CEA in solutions containing antibody modified HPGNP were evaluated by measuring the fluorescence quenching efficiency of pure CEA, CEA+BSA, CEA+AFP, CEA+IgG, and CEA+AFP. To remove the excess interfering proteins, the final mixed solution should be cleaned by centrifugation at a speed of 5000 rpm for 4 min. It should be noted that the nanoparticles are prone to aggregation after surface modification, therefore, the centrifugation speed and time should be precisely tuned. Besides, we can see that the addition of interfering compounds resulted in an only slight change in the quenching efficiency of CEA

distance is the critical parameter. Besides, as described by Silber and Kuhn (CPS-Kuhn) model,44−46 the distance which determines the fluorescence quenching or enhancing efficiency is size and shape dependent. Experimentally and theoretically, it has been evident that quenching efficiency becomes greater when the distance is decreased.25,47,48 Therefore, our experimental results have shown good consistency with these investigations. To study the relationship between the concentration of CEA and the intensity of fluorescence, the fluorescence quenching efficiency as a function of the concentration of CEA was plotted (Figure 9A). As indicated in Figure 9A, the degree of fluorescence quenching efficiency is in a CEA concentrationdependent manner. The experiments show that the increase of CEA level from 1 to 140 pg/mL results in a monotonous decrease of fluorescence quenching efficiency from 0.93 to 0.67. However, when the concentration is larger than 100 pg/mL, the rate of decreasing of the quenching efficiency becomes slower. Besides, a linear correlation between the fluorescence quenching efficiency and the level of CEA is obtained in the solution with the linear range from 2 to 100 pg/mL, and the detection limit of 1.5 pg/mL (calculated from seven independent measurements when the signal-to-noise ratio is 3) as shown in Figure 9B. In comparison with other spectroscopy methods, the fluorescence quenching methods based on fluorescence quenching effect of HPGNPs has competitive advantages due to the ultralow detection limit and the simple procedures as shown in Table 1. Although some Table 1. Performance Comparison of Different CEA Probes sensing method surface-enhanced Raman scattering surface-enhanced fluorescence plasmonic absorption resonance light scattering fluorescence quenching

LOD

detection range

ref

5 pg/mL

5 pg/mL−500 μg/mL

49

3 pg/mL

0.01−1 ng/mL

50

0.1 ng/mL 0.03 ng/mL

0.4−25 ng/mL 0.1−60 ng/mL

51 52

1.5 pg/mL

2−100 pg/mL

this work

methods have larger linear detection range, the detection limit is higher and the technological process is rather complicated. For example, Zhou et al. prepared a surface plasmon resonance biosensor with gold nanoparticles signal amplification and

Figure 10. (A) Selectivity of fluorescence probe for CEA detection against other potentially interfering proteins including BSA, AFP, IgG, and AFU. (B) Comparison of fluorescence quenching efficiency of different interfering proteins compounds such as CEA, CEA+BSA, CEA+AFP, CEA+IgG, and CEA+AFU in hollow porous gold nanoparticles. H


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(2) Burke, K. A.; Brenckle, M. A.; Kaplan, D. L.; Omenetto, F. G. Evaluation of the Spectral Response of Functionalized Silk Inverse Opals as Colorimetric Immunosensors. ACS Appl. Mater. Interfaces 2016, 8, 16218−16226. (3) Clark, M. F.; Adams, A. Characteristics of the Microplate Method of Enzyme-Linked Immunosorbent Assay for the Detection of Plant Viruses. J. Gen. Virol. 1977, 34, 475−483. (4) Park, Y.; Hanbicki, A.; Erwin, S.; Hellberg, C.; Sullivan, J.; Mattson, J.; Ambrose, T.; Wilson, A.; Spanos, G.; Jonker, B. A GroupIV Ferromagnetic Semiconductor: Mnxge1− X. Science 2002, 295, 651−654. (5) Wang, X.; Wang, C.; Qu, K.; Song, Y.; Ren, J.; Miyoshi, D.; Sugimoto, N.; Qu, X. Ultrasensitive and Selective Detection of a Prognostic Indicator in Early-Stage Cancer Using Graphene Oxide and Carbon Nanotubes. Adv. Funct. Mater. 2010, 20, 3967−3971. (6) Wu, X.; Xu, L.; Liu, L.; Ma, W.; Yin, H.; Kuang, H.; Wang, L.; Xu, C.; Kotov, N. A. Unexpected Chirality of Nanoparticle Dimers and Ultrasensitive Chiroplasmonic Bioanalysis. J. Am. Chem. Soc. 2013, 135, 18629−18636. (7) Hsieh, B.-Y.; Chang, Y.-F.; Ng, M.-Y.; Liu, W.-C.; Lin, C.-H.; Wu, H.-T.; Chou, C. Localized Surface Plasmon Coupled Fluorescence Fiber-Optic Biosensor with Gold Nanoparticles. Anal. Chem. 2007, 79, 3487−3493. (8) Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence. Chem. Rev. 2015, 115, 10530−10574. (9) Lin, D.; Wu, J.; Yan, F.; Deng, S.; Ju, H. Ultrasensitive Immunoassay of Protein Biomarker Based on Electrochemiluminescent Quenching of Quantum Dots by Hemin Bio-Bar-Coded Nanoparticle Tags. Anal. Chem. 2011, 83, 5214−5221. (10) Wang, Q.; Wang, W.; Lei, J.; Xu, N.; Gao, F.; Ju, H. Fluorescence Quenching of Carbon Nitride Nanosheet through Its Interaction with DNA for Versatile Fluorescence Sensing. Anal. Chem. 2013, 85, 12182−12188. (11) Rosi, N. L.; Mirkin, C. A. Nanostructures in Biodiagnostics. Chem. Rev. 2005, 105, 1547−1562. (12) Nagrath, S.; Sequist, L. V.; Maheswaran, S.; Bell, D. W.; Irimia, D.; Ulkus, L.; Smith, M. R.; Kwak, E. L.; Digumarthy, S.; Muzikansky, A.; et al. Isolation of Rare Circulating Tumour Cells in Cancer Patients by Microchip Technology. Nature 2007, 450, 1235−1239. (13) Giljohann, D. A.; Mirkin, C. A. Drivers of Biodiagnostic Development. Nature 2009, 462, 461−464. (14) Green, I. X.; Tang, W.; Neurock, M.; Yates, J. T. Spectroscopic Observation of Dual Catalytic Sites During Oxidation of Co on a Au/ Tio2 Catalyst. Science 2011, 333, 736−739. (15) Dubertret, B.; Calame, M.; Libchaber, A. J. Single-Mismatch Detection Using Gold-Quenched Fluorescent Oligonucleotides. Nat. Biotechnol. 2001, 19, 365−370. (16) Waddon, A. J.; Coughlin, E. B. Crystal Structure of Polyhedral Oligomeric Silsequioxane (Poss) Nano-Materials: A Study by X-Ray Diffraction and Electron Microscopy. Chem. Mater. 2003, 15, 4555− 4561. (17) Huang, C.-S.; George, S.; Lu, M.; Chaudhery, V.; Tan, R.; Zangar, R. C.; Cunningham, B. T. Application of Photonic Crystal Enhanced Fluorescence to Cancer Biomarker Microarrays. Anal. Chem. 2011, 83, 1425−1430. (18) Mayilo, S.; Ehlers, B.; Wunderlich, M.; Klar, T. A.; Josel, H.-P.; Heindl, D.; Nichtl, A.; Kürzinger, K.; Feldmann, J. Competitive Homogeneous Digoxigenin Immunoassay Based on Fluorescence Quenching by Gold Nanoparticles. Anal. Chim. Acta 2009, 646, 119− 122. (19) Zhu, J.; Wang, J.-F.; Li, J.-J.; Zhao, J.-W. Specific Detection of Carcinoembryonic Antigen Based on Fluorescence Quenching of AuAg Core-Shell Nanotriangle Probe. Sens. Actuators, B 2016, 233, 214− 222. (20) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins. Science 2003, 301, 1884−1886.

because of the competition in nonspecific binding. Hence, the presence of these proteins has minimal effect on the quantification of CEA using the fluorescence quenching method. In the experiments, the binding of the CEA antibody and antigen confers the specificity. As shown from the Figure 10, the results indicate the influence of other interfering proteins could be neglected in the detection, and the CEA could be selectively detected by using the CEA antibody modified novel hollow porous gold nanoparticles probe.

4. CONCLUSIONS We have prepared a novel CEA probe based on the fluorescence quenching efficiency of hollow porous gold nanoparticles. For the specific detection of CEA, the surface of the nanoparticles has been modified with CEA antibody. To optimize the fluorescence quenching efficiency and hence improve the sensitivity of hollow porous gold nanoparticles, we have systematically studied the nanoparticles parameters of particle size, surfactants, surface hole size, surface roughness, and the distance between the particles and the fluorescence molecules. The results show that all these parameters play important roles in determining the performance of CEA detection. Optimization of the parameters maximized the fluorescence quenching efficiency of hollow porous gold nanoparticles, and as a consequence improved the sensitivity of the probe. By using the probe, CEA can be detected with a detection limit of 1.5 pg/mL and a linear detection range from 2 to 100 pg/mL. The ultralow limit of detection and the simplicity in sample preparation provides competitive advantages in related spectroscopy methods. Furthermore, the selectivity of this probe has been evaluated by comparing to four interfering proteins including BSA, AFP, IgG and AFU. The results exhibit that the probe has high selectivity for CEA, and other proteins have negligible interference for the detection of CEA Therefore, this method enables selective detection of CEA at the ultralow level and therefore has great potential in the field of biomarker detection.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: 86-29-82664224. Fax: 86-29-82664224. E-mail: [email protected] (J.-J.L.). *Phone: 86-29-82664224. Fax: 86-29-82664224. E-mail: [email protected] (J.-W.Z.). ORCID

Jun-Wu Zhao: 0000-0003-4525-9094 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China under grant No. 61675162 and the Natural Science Basic Research Plan in Shaanxi Province of China under grant No. 2017JM6023 and 2017JM8064.

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REFERENCES

(1) Alizadeh, N.; Hallaj, R.; Salimi, A. A Highly Sensitive Electrochemical Immunosensor for Hepatitis B Virus Surface Antigen Detection Based on Hemin/G-Quadruplex Horseradish PeroxidaseMimicking Dnazyme-Signal Amplification. Biosens. Bioelectron. 2017, 94, 184−192. I


Research Article

ACS Applied Materials & Interfaces (21) Chen, Z.; Tabakman, S. M.; Goodwin, A. P.; Kattah, M. G.; Daranciang, D.; Wang, X.; Zhang, G.; Li, X.; Liu, Z.; Utz, P. J.; et al. Protein Microarrays with Carbon Nanotubes as Multicolor Raman Labels. Nat. Biotechnol. 2008, 26, 1285−1292. (22) Rodríguez-Lorenzo, L.; De La Rica, R.; Á lvarez-Puebla, R. A.; Liz-Marzán, L. M.; Stevens, M. M. Plasmonic Nanosensors with Inverse Sensitivity by Means of Enzyme-Guided Crystal Growth. Nat. Mater. 2012, 11, 604−607. (23) Daniel, M. C.; Astruc, D. Gold Nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications toward Biology, Catalysis, and Nanotechnology. Chem. Rev. 2004, 104, 293. (24) Link, S.; El-Sayed, M. A. Shape and Size Dependence of Radiative, Non-Radiative and Photothermal Properties of Gold Nanocrystals. Int. Rev. Phys. Chem. 2000, 19, 409−453. (25) Anger, P.; Bharadwaj, P.; Novotny, L. Enhancement and Quenching of Single-Molecule Fluorescence. Phys. Rev. Lett. 2006, 96, 113002. (26) Dulkeith, E.; Morteani, A.; Niedereichholz, T.; Klar, T.; Feldmann, J.; Levi, S.; Van Veggel, F.; Reinhoudt, D.; Möller, M.; Gittins, D. Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects. Phys. Rev. Lett. 2002, 89, 203002. (27) Gersten, J.; Nitzan, A. Spectroscopic Properties of Molecules Interacting with Small Dielectric Particles. J. Chem. Phys. 1981, 75, 1139−1152. (28) Lakowicz, J. R. Radiative Decay Engineering: Biophysical and Biomedical Applications. Anal. Biochem. 2001, 298, 1−24. (29) Leitner, A.; Lippitsch, M.; Draxler, S.; Riegler, M.; Aussenegg, F. Fluorescence Properties of Dyes Adsorbed to Silver Islands, Investigated by Picosecond Techniques. Appl. Phys. B: Photophys. Laser Chem. 1985, 36, 105−109. (30) Leung, P.; George, T. F. Molecular Fluorescence Spectroscopy in the Vicinity of a Microstructure. J. Chim. Phys. Phys.-Chim. Biol. 1995, 92, 226−247. (31) Kuo, C. H.; Chu, Y. T.; Song, Y. F.; Huang, M. H. Cu2o Nanocrystal-Templated Growth of Cu2s Nanocages with Encapsulated Au Nanoparticles and in-Situ Transmission X-Ray Microscopy Study. Adv. Funct. Mater. 2011, 21, 792−797. (32) Liu, J.; Guan, Z.; Lv, Z.; Jiang, X.; Yang, S.; Chen, A. Improving Sensitivity of Gold Nanoparticle Based Fluorescence Quenching and Colorimetric Aptasensor by Using Water Resuspended Gold Nanoparticle. Biosens. Bioelectron. 2014, 52, 265−270. (33) Xia, Y. N.; Li, W. Y.; Cobley, C. M.; Chen, J. Y.; Xia, X. H.; Zhang, Q.; Yang, M. X.; Cho, E. C.; Brown, P. K. Gold Nanocages: From Synthesis to Theranostic Applications. Acc. Chem. Res. 2011, 44, 914−924. (34) Xing, T.-Y.; Zhu, J.; Li, J.-J.; Zhao, J.-W. Morphology Modification of Gold Nanoparticles from Nanoshell to C-Shape: Improved Surface Enhanced Raman Scattering. J. Appl. Phys. 2016, 119, 243104. (35) Bastus, N. G.; Merkoci, F.; Piella, J.; Puntes, V. Synthesis of Highly Monodisperse Citrate-Stabilized Silver Nanoparticles of up to 200 Nm: Kinetic Control and Catalytic Properties. Chem. Mater. 2014, 26, 2836−2846. (36) Sun, Y. G.; Xia, Y. N. Mechanistic Study on the Replacement Reaction between Silver Nanostructures and Chloroauric Acid in Aqueous Medium. J. Am. Chem. Soc. 2004, 126, 3892−3901. (37) Lu, X.; Au, L.; McLellan, J.; Li, Z.-Y.; Marquez, M.; Xia, Y. Fabrication of Cubic Nanocages and Nanoframes by Dealloying Au/ Ag Alloy Nanoboxes with an Aqueous Etchant Based on Fe (No3) 3 or Nh4oh. Nano Lett. 2007, 7, 1764−1769. (38) He, Q.; Badu-Tawiah, A. K.; Chen, S.; Xiong, C.; Liu, H.; Zhou, Y.; Hou, J.; Zhang, N.; Li, Y.; Xie, X.; et al. In Situ Bioconjugation and Ambient Surface Modification Using Reactive Charged Droplets. Anal. Chem. 2015, 87, 3144−3148. (39) Rakshit, S.; Moulik, S. P.; Bhattacharya, S. C. Understanding the Effect of Size and Shape of Gold Nanomaterials on Nanometal Surface Energy Transfer. J. Colloid Interface Sci. 2017, 491, 349−357.

(40) Kerbellec, N.; Catala, L.; Daiguebonne, C.; Gloter, A.; Stephan, O.; Buenzli, J.-C.; Guillou, O.; Mallah, T. Luminescent Coordination Nanoparticles. New J. Chem. 2008, 32, 584−587. (41) Jiang, Y.; Yan, Y.-B.; Zhou, H.-M. Polyvinylpyrrolidone 40 Assists the Refolding of Bovine Carbonic Anhydrase B by Accelerating the Refolding of the First Molten Globule Intermediate. J. Biol. Chem. 2006, 281, 9058−9065. (42) Halik, M.; Klauk, H.; Zschieschang, U.; Schmid, G.; Ponomarenko, S.; Kirchmeyer, S.; Weber, W. Relationship between Molecular Structure and Electrical Performance of Oligothiophene Organic Thin Film Transistors. Adv. Mater. 2003, 15, 917−922. (43) Sun, S.; Gao, M.; Lei, G.; Zou, H.; Ma, J.; Huang, C. Visually Monitoring the Etching Process of Gold Nanoparticles by Ki/I2 at Single-Nanoparticle Level Using Scattered-Light Dark-Field Microscopic Imaging. Nano Res. 2016, 9, 1125−1134. (44) Kuhn, H. Classical Aspects of Energy Transfer in Molecular Systems. J. Chem. Phys. 1970, 53, 101−108. (45) Chance, R.; Prock, A.; Silbey, R. Comments on the Classical Theory of Energy Transfer. J. Chem. Phys. 1975, 62, 2245−2253. (46) Chance, R.; Prock, A.; Silbey, R. Molecular Fluorescence and Energy Transfer near Interfaces. Adv. Chem. Phys. 1978, 37, 65. (47) Liu, Y.; Loh, W. Q.; Ananthanarayanan, A.; Yang, C.; Chen, P.; Xu, C. Fluorescence Quenching between Unbonded Graphene Quantum Dots and Gold Nanoparticles Upon Simple Mixing. RSC Adv. 2014, 4, 35673−35677. (48) Samanta, A.; Zhou, Y.; Zou, S.; Yan, H.; Liu, Y. Fluorescence Quenching of Quantum Dots by Gold Nanoparticles: A Potential Long Range Spectroscopic Ruler. Nano Lett. 2014, 14, 5052−5057. (49) Chen, G.; Chen, Y.; Zheng, X.; He, C.; Lu, J.; Feng, S.; Chen, R.; Zeng, H. Surface-Enhanced Raman Scattering Study of Carcinoembryonic Antigen in Serum from Patients with Colorectal Cancers. Appl. Phys. B: Lasers Opt. 2013, 113, 597−602. (50) Yang, X.; Zhuo, Y.; Zhu, S.; Luo, Y.; Feng, Y.; Xu, Y. Selectively Assaying Cea Based on a Creative Strategy of Gold Nanoparticles Enhancing Silver Nanoclusters’ Fluorescence. Biosens. Bioelectron. 2015, 64, 345−351. (51) Špringer, T.; Homola, J. Biofunctionalized Gold Nanoparticles for Spr-Biosensor-Based Detection of Cea in Blood Plasma. Anal. Bioanal. Chem. 2012, 404, 2869−2875. (52) Chen, Z.; Lei, Y.; Liang, Z.; Li, F.; Liu, L.; Li, C.; Chen, F. A Highly Sensitive Label-Free Resonance Light Scattering Assay of Carcinoembryonic Antigen Based on Immune Complexes. Anal. Chim. Acta 2012, 747, 99−105.

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Specific Detection of Carcinoembryonic Antigen Based on

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