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Enhanced Fluorescence ELISA Based on HAT Triggering Fluorescence “Turn-on” with Enzyme−Antibody Dual Labeled AuNP Probes for Ultrasensitive Detection of AFP and HBsAg Yudong Wu,† Weisheng Guo,‡ Weipan Peng,† Qian Zhao,† Jiafang Piao,† Bo Zhang,† Xiaoli Wu,† Hanjie Wang,† Xiaoqun Gong,*,† and Jin Chang*,†
ACS Appl. Mater. Interfaces 2017.9:9369-9377. Downloaded from pubs.acs.org by STOCKHOLM UNIV on 01/19/19. For personal use only.
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School of Materials Science and Engineering, School of Life Sciences, Tianjin University and Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology (Tianjin), 92 Weijin Road, Nankai District, Tianjin 300072, P. R. China ‡ CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety National Center for Nanoscience and Technology, Beijing 100190, China S Supporting Information *
ABSTRACT: At present, enzyme-linked immunosorbent assay (ELISA) is considered to be the most appropriate approach in clinical biomarker detection, with good specificity, low cost, and straightforward readout. However, unsatisfactory sensitivity severely hampers its wide application in clinical diagnosis. Herein, we designed a new kind of enhanced fluorescence enzyme-linked immunosorbent assay (FELISA) based on the human alpha-thrombin (HAT) triggering fluorescence “turn-on” signals. In this system, detection antibodies (Ab2) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine110 with fluorescence quenched served as the substrate of HAT. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, leading to a strong fluorescence signal for sensing ultralow levels of alpha fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg). Under the optimized reaction conditions, AFP and HBsAg were detected at the ultralow concentrations of 10−8 ng mL−1 and 5 × 10−4 IU mL−1, respectively, which were at least 104 times lower than those of the conventional fluorescence assay and 106 times lower than those of the conventional ELISA. In addition, we further discussed the efficiency of the sensitive FELISA in clinical serum samples, showing great potential in practical applications. KEYWORDS: fluorescence enzyme-linked immunosorbent assay, gold nanoparticles, human alpha-thrombin, alpha fetoprotein, hepatitis B virus surface antigen
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INTRODUCTION Recently, detection of biomarkers with ultrahigh sensitivity gained intense attention due to its enormous function in early stage disease diagnosis, biomarker content monitoring, and therapeutic information feedback.1−4 It has been reported that early diagnosis may help to improve therapeutic intervention.5 As known to all, ELISA was the most popular immunoassay in clinical biomarkers detection, due to its good specificity, low cost, and straightforward readouts.6,7 However, the sensitivity of conventional ELISA was powerless to screen ultralow concentration of biomarkers in some diseases early stages.8,9 Thus, there was an urgent need to develop ultrasensitive detection methods for different kinds of biomarkers. At present, the fluorescence immunoassay, showing good compatibility with the currently available analytical platforms, is considered to be one of the most sensitive approaches for screening biomarkers.10−14 Hence, the marriage of ELISA and the fluorescence immunoassay showed exciting clinical diagnosis © 2017 American Chemical Society
potential due to the complementary diagnostic capacity, known as FELISA. In the past few decades, many efforts focused on the promising FELISA, including horse radish peroxidase (HRP)based chemiluminescent immunoassays,15 alkaline phosphatase (ALP)-based chemiluminescent immunoassays,16 enhanced luminescence enzyme immunoassay,17−19 and so on. However, the fundamental limitations of fluorescence immunoassays, such as serious fluorescence quenching and unsatisfactory sensitivity, severely hampered its application in clinical diagnosis. In other words, the sensitivity of these conventional fluorescence methods still cannot satisfy the ultrasensitive detection requirements. Now, one of the most effective approaches to improve the sensitivity of FELISA is searching for a highly efficient enzyme Received: December 18, 2016 Accepted: March 2, 2017 Published: March 2, 2017 9369
DOI: 10.1021/acsami.6b16236 ACS Appl. Mater. Interfaces 2017, 9, 9369−9377
Research Article
ACS Applied Materials & Interfaces Scheme 1. Schematic Diagram of the Enhanced FELISA for the Detection of AFP and HBsAga
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(a) Procedures of the proposed immunoassays with the newly dual labeled AuNP probes multi-HAT-AuNP-Ab2; (b) Enzymatic hydrolysis of the bisamide Rhodamine110 substrates. Technologies Inc. (Essex Junction, VT). Tetrachloroauric acid trihydrate (HAuCl4), glutaraldehyde, and trisodium citrate were supplied by Sigma. Mouse monoclonal primary antihuman AFP antibody, secondary antihuman AFP antibody, AFP antigen, Anti-HBsAg antibody, and HBsAg were purchased from Bioscience Diagnostic Technology Co. (Tianjin, China). The 96-well polystyrene plates were obtained from Corning Costar. Tween-20 was purchased from Alfa Aesar. Bovine serum albumin (BSA), fetal bovine serum (FBS), lysine hydrochloride, and polyethylene glycol (PEG, number-average molecular weight (Mn) = 20000) were supplied by DG Biotechnology Co. (Beijing, China). Sodium periodate, ammonium chloride, sodium boron hydride, potassium carbonate, nitric acid, and hydrochloric acid were supplied by JT Chemical Reagent (Tianjin, China). The serum samples were collected from Tianjin Medical University General Hospital. The deionized water used in this work has a resistivity of 18.2 MΩ·cm, which was of Milli-Q grade. The ultraviolet−visible spectrum of AuNPs were measured by an ultraviolet−visible spectrophotometer (PerkinElmer, United States). TEM images (size and morphology) of all AuNPs were recorded with an analytical electron microscope. We recorded dynamic light scattering (DLS) with a zeta potential analyzer (BrookHaven Instruments Corporation). The fluorescence intensity in the 96-well polystyrene plate was detected by an EnSpire Multilabel Reader (PerkinElmer, United States) at 521 nm. The fluorescence images of Rhodamine110 was monitored with a signal acquisition device, Azure C600 (Azure Biosystems, Inc.). Synthesis and Characterization of AuNPs. All glass services were immersed in aqua regia, washed three time with deionized water, and stored at 150 °C. AuNPs were prepared according to the previous reports.29−31 To prepare smaller-sized AuNPs (
