LETTER pubs.acs.org/Langmuir
Gold Nanoparticle Layer: A Promising Platform for Ultra-Sensitive Cancer Detection Feng Zhou,†,‡ Lin Yuan,*,† Hongwei Wang,† Dan Li,†,‡ and Hong Chen*,† †
College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199# Ren’ai Road, Suzhou 215123, P. R. China ‡ School of Materials Science and Engineering, Wuhan University of Technology, Wuhan 430070, P. R. China
bS Supporting Information ABSTRACT:
Developing new technologies applicable to the sensitive detection of cancer in its early stages has always been attractive in diagnosis. A stable gold nanoparticle layer (GNPL)-modified high-binding ELISA plate was obtained via chemical plating and was proven to be more efficient in binding proteins while maintaining their activity. GNPL-based ELISA for the representative biomarker carcinoembryonic antigen (CEA) demonstrated that GNPL markedly amplified the ELISA signal and significantly improved the limit of detection (LOD). Antithrombin detection further confirms the effectiveness and universality of this GNPL-based platform. The entire assay procedure is simple and low in cost and does not require special facilities. All these virtues indicate that this GNPL platform holds great promise in clinical applications for the early diagnosis of cancer.
’ INTRODUCTION Diagnosis and treatment of cancer in its nascent stages are of great importance because of the widespread occurrence of the disease, its high death rate, and the frequency of recurrence after treatment. Moreover, the survival rate of cancer patients is heavily dependent on early diagnosis.1 Hence, developing technologies applicable for the sensitive detection of cancer at the initial stages has always been one of the most pressing issues in diagnosis.2 Tests for representative biomarkers are routinely employed for early cancer screening and to monitor the effectiveness of cancer treatments.3,4 However, the concentration of these biomarkers in vivo is usually too low to be detected via the present routine diagnostic methods at the initial stage of specific cancers. Therefore, decreasing the limit of detection (LOD) is vitally important for clinical diagnosis. Gold nanoparticles and the nanostructures they constitute hold great promise in diagnostic fields due to their unique properties, such as their large surface area-to-volume ratio, high loading capacity, stability, and efficient electron transfer.5 All these advantages make gold-nanostructured materials widely r 2011 American Chemical Society
applicable in novel diagnostic devices, such as various chemical and biological sensors,6-9 surface enhanced Raman spectroscopy (SERS),10,11 and DNA/protein arrays.12,13 Though promising results have been achieved, the fabrication process of these nanostructured materials is not always easy, and the high cost of the sophisticated facilities required for those assays also restricts their popularization. Enzyme-linked immunosorbent assay (ELISA) is probably one of the most widely adopted techniques in clinical diagnosis owing to its simplicity, low cost, readability, acceptability, and sensitivity.14,15 However, this microplate surface-based assay also has intrinsic drawbacks, such as the low binding capability of antigens or antibodies on the surface. The adsorbed proteins are less accessible for the ensuing antigen-antibody recognition and binding when displayed on a two-dimensional microplate surface, thus lowering the binding efficiency.16 In addition, the Received: December 16, 2010 Revised: January 28, 2011 Published: February 14, 2011 2155
dx.doi.org/10.1021/la1049937 | Langmuir 2011, 27, 2155–2158
Langmuir
LETTER
Figure 1. Characterization of pristine (control) and GNPL-modified ELISA plate. The volume of the plating solution for GNPL a-e is as follows: 50, 100, 150, 200, and 250 μL, respectively. a1-e1 are the light micrographs (bar: 100 μm); a2-e2 are micrographs of field emission scanning eletron microscopy (FESEM) (bar: 10 μm); a3-e3 are amplified micrographs of FESEM (bar: 100 nm); the bottom figures are the respective porosity values for GNPL a-e.
ultimate limit of detection of ELISA is usually not low enough to meet the challenges of biomarker detection of cancer at the initial stages.14,15 Therefore, many groups have worked to improve the ELISA for better performance in biomarker detection.14,15,17,18 In this research, we find that the facilely fabricated threedimensional gold nanoparticle layer (GNPL)-decorated ELISA plate significantly amplifies the ELISA signal and decreases the LOD in the detection of carcinoembryonic antigen (CEA), an important biomarker closely related to many cancers.19 This demonstrates that GNPL-based ELISA could be a powerful tool for the early diagnosis of cancer not only because of its remarkable sensitivity, but also because it is low in cost, easy to prepare, and does not alter the flow of conventional ELISA. All these virtues make it a promising candidate for superior conventional ELISA plate substitute in practical use. Moreover, we also find that our method is applicable to the fabrication of GNPL on common material surfaces, which further broadens the scope of potential applications of this GNPL platform in other fields, such as nucleic acid and protein microchips, high-throughput catalytic surfaces, and more.
’ RESULTS AND DISCUSSION The stable GNPL on a commercial high-binding ELISA plate was prepared via a chemical plating method with some minor modifications.20 By adjusting the amount (50-250 μL) of plating solution into each well, a series of GNPLs (a-e) of different porosity rates was obtained and the morphology was characterized. According to light microscopy, the intensity of the reflected light on GNPL a-e decreased gradually (Figure 1a1e1) as the amount of plating solution increased, which is indicative of the morphological differences. Field emission scanning electron microscopy (FESEM) (Figure 1a2-e2 and a3-e3) shows that the GNPLs are actually three-dimensional micro- and nanosized porous structures, which consist of gold nanoparticles with sizes ranging from 100 to 150 nm. These structures became more densely aggregated with the increment of plating solution. ImageJ (public software from National Institutes of Health; http://rsbweb.nih.gov/ij/) was used to analyze the porosity value.21 The result shows that the porosity value decreased gradually with the increment of plating solution (n = 3).
As the micro- and nanostructured surfaces are more efficient in capturing biomolecules due to their large surface area-to-volume ratio,16,22 we compared the adsorption of lysozyme (LYZ), human serum albumin (HSA), and fibrinogen (Fg)—three model proteins of different sizes on GNPL a-e and pristine high-binding ELISA plates (n = 3). It can be clearly seen from Figure 2a that the amount of protein adsorbed on GNPL a-e increased noticeably; e.g., for GNPL e, the amounts of adsorbed LYZ, HSA, and Fg are 2.76-, 2.34-, and 3.26fold higher than that on pristine high-binding ELISA plate, respectively. Therefore, it can be expected that the binding density of proteins on GNPL-modified surfaces is largely increased, and the high binding density of proteins on the surface is especially meaningful for the enhancement of the detection ability of ELISA.16,22 On the other hand, it is reported that surfaces with micro- and nanostructured surfaces may alter the conformation of the adsorbed proteins and subsequently affect the protein activity.23-26 Hence, we compared the effect of GNPL a and e on the activity of adsorbed LYZ (n = 3). As seen in Figure 2b, the activity of LYZ adsorbed on GNPL a and e is 2.65- and 3.86-fold higher than that on a high-binding ELISA plate, respectively. We speculated that the superior activity could be attributed to the micro- and nanosized three-dimensional structures that not only are more efficient in adsorbing LYZ, but also facilitate the binding process toward LYZ substrate molecules compared to the twodimensional surface of the high-binding ELISA plate. This could be attributed to both the altered molecular conformation and less steric hindrance on the three-dimensional micro- and nanostructured surfaces.16,22,23 Fortunately, the high binding density and activity maintenance of adsorbed proteins are the exact requirements for an ideal ELISA plate. Therefore, to prove our hypothesis, we chose GNPL e as the substrate in the subsequent CEA detection because it possessed the largest protein-binding ability and activity preservation. CEA, a widely used cancer biomarker, was chosen as a target molecule in our indirect format of ELISA. The normal range of CEA in vivo is
