Metal-Linked Immunosorbent Assay (Melisa): the Enzyme-Free

Theranostics 2016, Vol. 6, Issue 10 1732 Ivyspring International Publisher Theranostics 2016; 6(10): 1732-1739. doi: 10.7150/thno.16129 Research Paper Metal-linked Immunosorbent Assay (MeLISA): the Enzyme-Free Alternative to ELISA for Biomarker Detection in Serum Ru-Jia Yu1†, Wei Ma1†, Xiao-Yuan Liu1, Hong-Ying Jin1, Huan-Xing Han2, Hong-Yang Wang3, He Tian1, Yi-Tao Long1 1. Key Laboratory for Advanced Materials, School of Chemistry & Molecular Engineering, East China University of Science and Technology, P. R. China. 2. Translational Medicine Center, Changzheng Hospital, The Second Military Medical University, P. R. China. 3. Laboratory of Signal Transduction, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, P. R. China. †These authors contributed equally. Corresponding authors: H.T. ([email protected]) or Y.T.L. ([email protected]) © Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions. Received: 2016.05.11; Accepted: 2016.05.31; Published: 2016.06.27 Abstract Determination of disease biomarkers in clinical samples is of crucial significance for disease monitoring and public health. The dominating format is enzyme-linked immunosorbent assay (ELISA), which subtly exploits both the antigen-antibody reaction and biocatalytic property of enzymes. Although enzymes play an important role in this platform, they generally suffer from inferior stability and less tolerant of temperature, pH condition compared with general chemical product. Here, we demonstrate a metal-linked immunosorbent assay (MeLISA) based on a robust signal amplification mechanism that faithfully replaces the essential element of the enzyme. As an enzyme-free alternative to ELISA, this methodology works by the detection of α-fetoprotein (AFP), prostatic specific antigen (PSA) and C-reactive protein (CRP) at concentrations of 0.1 ng mL-1, 0.1 ng mL-1 and 1 ng mL-1 respectively. It exhibits approximately two magnitudes higher sensitivity and is 4 times faster for chromogenic reaction than ELISA. The detection of AFP and PSA was further confirmed by over a hundred serum samples from hepatocellular carcinoma (HCC) and prostate cancer patients respectively. Key words: MeLISA Introduction Enzyme-free detection is extremely desirable The development of nanotechnology opens the [1–3] yet still challenging to the engineering of robust way for advances in scientific research [12–18]. With methods which would renovate immunoassay and the synthetic procedure towards nanoparticles having improve current diagnostic techniques. Although been optimized, biosensors based on nanomaterials extensive research has been devoted including the use are moving into clinical practice and commercial of electrochemistry [4–6], fluorescence [7,8] and translation [19]. Conventional gold nanoparticles chemiluminescence [9–11], the requirement of (AuNPs)-based colorimetric detection yields high additional expensive equipment discourages their sensitivity due to the high extinction coefficient and broad application especially in resource limited distance-dependent optical properties [20–25], regions. Since the color generation is the basis of nonetheless, current AuNPs aggregation induced by ELISA, engineering enzyme-free colorimetric assay interparticle crosslinking of biomolecules often could not only well accommodate conventional requires careful and long-time incubation in a buffer immunoassay, but also greatly improve its stability. solution which may cause undesirable aggregation of http://www.thno.org Theranostics 2016, Vol. 6, Issue 10 1733 AuNPs [26]. Robust, ultra-rapid and highly stable Alkynyl silvers induced colorimetric AuNPs aggregation in colorimetric assay still remains assay a challenge. We circumvent this problem by presenting an organic reaction-induced aggregation The formation of alkynyl silvers has wide in aqueous solution which occurs within 1 second. applications in organic synthesis [27,28], This aggregation of AuNPs is triggered by the silver-catalyzed reaction [29,30] and building blocks reaction between terminal alkynes and silver ions, for complex architectures [31,32]. To employ this which has been used as a “traditional test” for organic reaction for colorimetric assay, terminal acetylenic groups (depicted in Fig. 1). We adopt this alkyne-functionalized thiol (5-(1,2-dithiolan-3-yl)-N- classic reaction for the first time in a colorimetric (prop-2-ynyl) pentanamide) was synthesized assay. Surprisingly, it yielded the fastest following previously reported methodology [33]. This AuNPs-based colorimetric process ever reported compound was spontaneously self-assembled onto (Table S1). the surface of AuNPs through Au-S bonds which Herein, this colorimetric assay was integrated could be confirmed by a slight red shift in the UV-Vis into immunoassay by employing the silver ions spectra (Fig. S1). By introducing silver ions to the generation amplification. In our approach, silver alkyne-functionalized AuNPs, the H of the C-H bond nanoparticles (AgNPs) were oxidatively dissolved in of the alkyne was exchanged by the silver (I) species, the presence of hydrogen peroxide to produce immediately forming alkynyl silvers complex. Fig. 2a millions of silver ions. Instead of the standard shows the different tonality of the enzymatic reaction under well-defined conditions in alkyne-functionalized AuNPs solution upon an ELISA test, the robust chemical dissolution and the treatment with different concentrations of silver ultra-rapid colorimetric process ensure a high nitrate; the colour intensity increased at higher reproducible and practical system. The amplification concentrations of silver ions, and the colour remained mechanism exhibits attractive size-modulation, that fixed overnight. The blue coloured solutions were is, AgNPs with differing diameters generate silver characterized by the UV-Vis spectra as demonstrated ions of corresponding amounts, leading to a in Fig. 2b. At higher concentration of silver ions, the controlled detecting range of analyte. That is of vital absorbance at 620 nm (A620 nm) was intensified, which importance for an analytical methodology since the was related to aggregation of AuNPs, whereas the reference ranges for different analytes varies extinction at 520 nm (A520nm), corresponding to significantly. The MeLISA offers a simple but effective dispersed AuNPs, was decreased. The absorbance platform for biomarker detection with high stability ratio between A620 nm and A520 nm was proportional to and robustness. the concentration of silver ions below 10 μM (Fig. 2c), indicating that this colorimetric assay was potentially quantifiable. Figure 1. Schematic representation of the MeLISA for the detection of biomarker. In this sandwich immunoassay, the polystyrene plate is covered with a capture antibody which could anchor the target biomarker. This coating step comprises the blocking procedure with bovine serum albumin to minimize nonspecific interactions. Serum sample with target antigen is added to the plate to allow the reaction between capture antibody and target antigen. Finally, the sandwich immunoassay is formed by the addition of the detection antibody that conjugated with silver nanoparticles. Introduction of hydrogen peroxide initiates the dissolution of AgNPs into silver ions, leading to a rapid aggregation of alkyne-functionalized AuNPs. The aggregation yields a distinct red-to-blue colour change in solution that can be read by eye or a compatible plate reader (for quantification). http://www.thno.org Theranostics 2016, Vol. 6, Issue 10 1734 Figure 2. The aggregation of alkyne-functionalized AuNPs induced by silver ions. a, Visual colour distribution upon treatment of the alkyne-functionalized AuNPs system with various concentrations of Ag+ range from 1 to 400 μM. The photograph was taken less than three seconds after the addition of Ag+. b, UV-Vis spectra of the alkyne-functionalized AuNPs system in the presence of Ag+ with concentrations from 1 to 400 μM. The absorbance at 620 nm indicates the aggregation of alkyne-functionalized AuNPs. c, Plot of A620 nm/A520 nm against different concentrations of Ag+. The inset is a linear relationship at low concentration of Ag+. Error bars are based on three independent measurements. d, e, Transmission electron microscopy (TEM) images of the dispersed and aggregated alkyne-functionalize AuNPs in the absence (d) and presence (e) of Ag+. Scale bar: 200 nm. f, DLS analysis of alkyne-functionalized AuNPs in the presence of 0 μM (gray), 4 μM (blue), 8 μM (orange), 40 μM (purple) silver ions. The aggregation was due to the formation of compound. Taken together, the aggregation of alkynyl silvers which could change the surface charge alkyne-functionalized AuNPs induced by silver ions of alkyne-functionalized AuNPs. The high efficient can be used in colorimetric assay due to the rapid nature of this organic reaction, results in a fast response, high stability and capability to produce an aggregation within 1 second at room temperature. intense optical response. This is the most rapid AuNPs-based colorimetric process ever reported. The aggregation of MeLISA for biomarker detection alkyne-functionalized AuNPs could be directly We used the dissolution of AgNPs for signal confirmed by transmission electron microscopy amplification, taking the place of enzymatic process. (TEM) images. It clearly displayed a dispersed

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