Surface Modification of Gold Nanoparticles with Small Molecules for

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Surface Modification of Gold Nanoparticles with Small Molecules for Biochemical Analysis Yiping Chen,†,# Yunlei Xianyu,†,# and Xingyu Jiang*,†,‡ †

Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, 11 BeiYiTiao, ZhongGuanCun, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China CONSPECTUS: As one of the major tools for and by chemical science, biochemical analysis is becoming increasingly important in fields like clinical diagnosis, food safety, environmental monitoring, and the development of chemistry and biochemistry. The advancement of nanotechnology boosts the development of analytical chemistry, particularly the nanoparticle (NP)-based approaches for biochemical assays. Functional NPs can greatly improve the performance of biochemical analysis because they can accelerate signal transduction, enhance the signal intensity, and enable convenient signal readout due to their unique physical and chemical properties. Surface chemistry is a widely used tool to functionalize NPs, and the strategy for surface modification is of great significance to the application of NP-mediated biochemical assays. Surface chemistry not only affects the quality of NPs (stability, monodispersity, and biocompatibility) but also provides functional groups (−COO−, −NH3+, −CHO, and so on) or charges that can be exploited for bioconjugation or ligand exchange. Surface chemistry also dictates the sensitivity and specificity of the NP-mediated biochemical assays, since it is vital to the orientation, accessibility, and bioactivity of the functionalized ligands on the NPs. In this Account, we will focus on surface chemistry for functionalization of gold nanoparticles (AuNPs) with small organic molecules for biochemical analysis. Compared to other NPs, AuNPs have many merits including controllable synthesis, easy surface modification and high molar absorption coefficient, making them ideal probes for biochemical assays. Small-molecule functionalized AuNPs are widely employed to develop sensors for biochemical analysis, considering that small molecules, such as amino acids and sulfhydryl compounds, are more easily and controllably bioconjugated to the surface of AuNPs than biomacromolecules due to their less complex structure and steric hindrance. The orientation and accessibility of small molecules on AuNPs in most cases can be precisely controlled without compromising their bioactivity as well, thus ensuring the performance, such as the specificity and sensitivity, of AuNP-based biochemical assays. This Account reviews recent progress in the surface chemistry of functionalized AuNPs for biochemical assays. The surface chemistries mainly include click chemistry, ligand exchange reaction, and coordination-based recognition. These surface-modified AuNPs allow for assaying a range of important biochemical markers including metal ions, small biomolecules, enzymes, and antigens and antibodies. Applications of these systems range from environmental monitoring to medical diagnostics. This Account highlights the advantages and limitations (sensitivity, detection efficiency, and stability) that AuNP-mediated assays still have compared with conventional analytical methods. This Account also discusses the future research directions of surfacemodified AuNP-mediated biochemical analysis. The main aim of this Account is to summarize the current surface modification strategies for AuNPs and further demonstrate how to make use of surface modification strategies to effectively improve the performance of AuNP-mediated analytical methods for a wide variety of applications relating to biochemical analysis. signal readout6 due to their unique physical and chemical properties such as large surface-to-volume ratio7 and excellent optical,8 electrical,9 magnetic, and catalytic properties.10 Functional NPs can accelerate signal transduction, enhance the signal intensity, and enable convenient signal readout; thus they can greatly improve the detection efficiency and broaden the applications of assays.11 Surface chemistry is a widely used tool to functionalize NPs, and the strategy for surface modification is vital to the application of NP-mediated

1. INTRODUCTION Biochemical analysis is becoming increasingly important in chemical science because of its broad applications in not only analytical chemistry1,2 but also organic synthesis, environmental chemistry, physical chemistry, and so forth. A successful sensing methodology essentially depends on the following four aspects: (1) a simple sample pretreatment; (2) a specific recognition element; (3) an effective signal transduction or amplification,3 and (4) a straightforward signal readout.4 Functional nanoparticles (NPs) have been widely used to develop sensing approaches because they can act as the carriers of sample pretreatment,5 signal recognition elements, or the probes for © 2017 American Chemical Society

Received: October 6, 2016 Published: January 9, 2017 310

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Accounts of Chemical Research biochemical assays.12 Surface chemistry not only affects the quality of NPs (stability, monodispersity, and biocompatibility) but also provides functional groups (−COO−, −NH3+, −CHO, and so on) or charges that can be exploited for the bioconjugation or ligand exchange.13 In addition, surface chemistry is of great significance to the orientation, accessibility, and bioactivity of the targeting molecule that dictates the sensitivity and specificity of the biochemical assays.14 For certain types of application, NPs may be functionalized with a wide range of surface chemistry to prepare functional nanoprobes that enable target recognition, effective signal amplification, and straightforward signal readout.15 AuNPs exhibit unique optical properties that depend on their size, shape, and morphology, and AuNP solution shows different colors in a shape−size-dependent manner.16 Targetinduced aggregation of AuNPs (typically 10−50 nm in diameter) will lead to a visible color change of solution from red to blue, owing to the interparticle plasmon coupling that results in an absorption band shift in the visible region of the electromagnetic spectrum. The color change of AuNPs in solution depends on the concentration of targets, which provides an excellent platform to design visible sensors for biochemical analysis of a variety of analytes. Compared to other NPs, AuNPs have several advantages including controllable synthesis,17 easy surface modification,18 and high molar absorption coefficient, making AuNPs ideal nanoprobes for sensing.19 In addition, AuNPs have good biocompatibility and low toxicity, being excellent candidates for in vivo imaging and antibacterial agents. Among all the AuNP-based biosensors, a bioanalysis strategy that employs the color change of AuNPs for naked-eye readout is the most popular. The state change of AuNPs (dispersed to aggregated or vice versa) will affect the color of AuNPs, which allows straightforward readout without any equipment,20 which has the potential for point-of-care testing (POCT).21 AuNP-mediated biochemical analysis has been developed for a variety of practical applications.22 A number of articles have reviewed the AuNP-mediated assays for detection of metal ions, nucleic acids, and proteins.23,24 Surface modification of AuNPs with polymers or biomacromolecules such as proteins and nucleic acids for biological applications is also discussed in other reviews.25 However, few reviews focused on the strategies for surface functionalization of AuNPs with small molecules for biosensing applications.26 Small molecules, such as amino acids and sulfhydryl compounds, are more easily and controllably bioconjugated to the surface of AuNPs than biomacromolecules such as antibody, enzyme, or polysaccharide due to the less complex structure and steric hindrance of small molecules. In addition, the orientation and accessibility of small molecules can be precisely controlled without compromising their bioactivity; thus small-molecule functionalized AuNPs might be more favorable for the stability and repeatability in detection of biochemical assays. Since the surface chemistry of AuNPs is significant to biochemical assays, it is necessary to summarize the currently used strategies for surface modification of AuNPs with small molecules and discuss the challenges and perspectives related to it. Thus, this Account focuses on recent progress in the surface chemistry for functionalization of AuNPs with small molecules for biochemical assays, including click chemistry, ligand exchange strategy, and coordination-based recognition (Scheme 1). In this Account, the strategies for surface modification of AuNPs are discussed, as well as their indispensable roles in AuNPmediated biosensors.

Scheme 1. Strategies of Surface Chemistry for Functionalization of AuNPs with Small Molecules and Their Application in Biochemical Analysis

2. SURFACE CHEMISTRY OF AuNPs FOR ASSAYS 2.1. Click Chemistry-Based Surface Conjugation of AuNPs

Click chemistry is extensively used for conjugation in materials science and biology, including the synthesis of polymers,27 bioconjugation,28,29 and imaging applications,30 but we have mainly used the components of click chemistry for analytical purposes. Click chemistry is an excellent strategy for surface modification because it does not affect the structure of NPs.31 Among different types of click reaction, Cu(I)-catalyzed 1,3dipolar cycloaddition of azides and alkynes (CuAAC) is the most widely used reaction.32 Without Cu(I), the reaction between azides and alkynes only proceeds at high temperatures or very slowly at room temperature.33 When catalyzed by Cu(I), the reaction speeds up in aqueous solutions at room temperature. We directly used the components of CuAAC for biochemical analysis, and we modified the AuNPs using thiolterminated azide and thiol-terminated alkyne to develop a series of CuAAC-mediated assays that use AuNPs for naked-eye readout. In this section, we focus on the progress of CuACCmodified AuNPs for biochemical analysis. Based on the color change of AuNPs (from red to blue) and the high selectivity of CuAAC, we modified the AuNPs using CuAAC for detecting Cu(II).34 AuNPs were modified with thiol-azide and thiol-alkyne by the Au−S bond, and CuAAC reaction can cross-link the azide and alkyne to result in the aggregation of AuNPs. This aggregation leads to the color change of AuNPs from red to blue, and the degree of aggregation depends on the concentration of Cu(I) in samples. This assay can be employed for Cu(II) detection by reducing Cu(II) into Cu(I) (Figure 1A). The conversion of Cu(II) into Cu(I) is quantitative when sodium ascorbate is used as a reductant, and this reaction takes place within seconds. The biggest advantage of this method is that combining the specificity of click chemistry and the outstanding optical property of AuNPs realizes detection of Cu(II) with high 311

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Figure 1. CuAAC-mediated surface chemistry of AuNPs for visual detection of Cu(II) and proteins. (A) Azide- and alkyne-terminated ligands are used to modify the AuNPs, and these functionalized AuNPs can be triggered to aggregate in the presence of Cu(II) by CuAAC. Adapted with permission from ref 34. Copyright 2008 Wiley. (B) CuAAC-mediated surface modification of AuNPs for quantifying protein..

sensitivity (limit of detection, LOD = 50 μM) and selectivity by naked eyes without any instrument. On the basis of this work, many works based on AuNPs and CuAAC have been developed for highly sensitiveand selective naked-eye detection of Cu(II) and related reducing agents. Because a reducing agent is required to reduce Cu(II) to Cu(I) for CuAAC, CuAAC-mediated AuNPs assays can be employed to detect reducing agents. Proteins as a reducing agent can reduce Cu(II) to Cu(I) because of the reducing nature of the peptide bond. A CuAAC-mediated strategy thus can be exploited to determine the concentration of the total proteins (in g/mL) in samples35 (Figure 1B). The linear range of detection (30−2500 μg mL−1) is broader than that of conventional bicinchoninic acid (BCA) and Bradford assays. In addition, this approach can tolerate most detergents and illegal additives such as melamine, which makes it a promising tool for detection of the total amount of protein in complicated samples, such as whole milk. We also developed a series of CuAAC-mediated immunoassays using functional AuNPs for the detection of many types of disease biomarkers. Because a large repertoire of antibodies (that specifically bind proteins, nucleic acids, and small molecules) are available, enzyme-linked immunosorbent assay (ELISA) has been widely applied for detecting disease biomarkers, the control of food quality, and the monitoring of environment. Even though traditional ELISA is powerful, its drawbacks are also quite obvious, such as instrument-dependence, high cost, and lengthy time. To solve these problems, we used copper oxide nanoparticle (CuO NP)-labeled antibody to replace conventional enzyme-labeled antibody and develop a more robust immunoassay, particularly for naked eye readout.36 The concentration of Cu(II) released from CuO NP-labeled antibodies reflected the amount of target in the samples by the immunoreaction. Accordingly, the degree of aggregation of AuNPs caused by the CuACC relates to the concentration of target that can be recognized through the antigen−antibody interaction (Figure 2A). This method has been successfully used to detect human immunodeficiency virus antibody in real

Figure 2. CuAAC-mediated surface modification of AuNPs for immunoassays. (A) Immunoassays based on the CuO-labled antibody and azide- and alkyne-modified AuNPs. CuO NPs are dissolved to release copper ions that trigger the aggregation of the AuNPs via CuAAC. Adapted with permission from ref 36. Copyright 2011 Wiley. (B) Immunoassay based on ALP-triggered CuAAC between azide- and alkyne-functionalized AuNPs.

serum samples with an accuracy of 100%. The biggest advantage of this method is the CuO NP-labeled antibody instead of the enzyme as the signal producer, which can be cheaper and more robust than the biological enzyme for mass 312

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Figure 3. Amino acid-mediated surface modification of AuNPs for biochemical analysis. (A) A colorimetric logic system (AND + OR) using arginine/lysine-functionalized AuNPs. (B) Microfluidic platform combines with molecular logic gates for detection of metal ions. Panels 1 and 2 are schematic illustration and photograph of the microfluidic chip. Panels 3 and 4 are TEM images of the AuNPs and the corresponding color of the observation window. Adapted with permission from ref 40. Copyright 2014 Wiley.

Figure 4. Arginine-modified AuNPs for GSH sensing in cancer cell using a dispersion-dominated chromogenic strategy. Adapted with permission from ref 41. Copyright 2015 Wiley.

and the color change of AuNPs can reflect the amount of target in the sample. Compared with conventional ELISA, this CuAAC-based AuNP-implemented immunoassay has a higher sensitivity for naked-eye detection owing to the “enzyme” amplification and “click” amplification. This assay has been successfully employed to detect the serum of patients who suffer from infection by Mycoplasma pneumoniae (MP) with an accuracy of 100%, while that of conventional ELISA is only 50%. Compared to the immunoassay based on CuO NPmodified antibody, this assay is more easily adaptable to existing immunoassays, because neither the antigen nor the antibody requires direct modification with the nanomaterials. The broad availability of ALP-labeled antibodies allows this assay to become useful for the naked-eye readout of essentially all immunoassays.

production. However, a main problem of this method is that the conjugation of antibody to CuO NPs might affect the activity of the antibody. Since the exact locale on the antibody for conjugation is not definitive, the possibility of CuO binding to the antibody-binding region (Fab) and thus affecting the effectiveness of the labeled antibody cannot be excluded. To address the problem of attenuated antibody activity by CuO NP-modification, we developed an immunoassay based on commercially available alkaline phosphatase (ALP)-labeled antibody and CuAAC-mediated strategy that enables sensitive and straightforward naked-eye detection37(Figure 2B). The ALP-labeled secondary antibody replaces CuO NP-labeled secondary antibody in this strategy to accommodate conventional ELISA because ALP is one of the most broadly used labeling enzyme in immunoassays. ALP-conjugated secondary antibodies are widely provided by suppliers for labeled antibodies, and this conjugate allows the site-specific modification of the antibody such that the labeling ALP does not affect the Fab region of the antibody. Under ALP-catalyzed dephosphorylation, the nonreducing ascorbic acid-phosphate can be transformed into ascorbic acid, which reduces Cu(II) to Cu(I). CuAAC reaction results in the aggregation of AuNPs,

2.2. Surface Modification of AuNPs Using Coordination-Based Recognition

2.2.1. Amino Acid-Mediated Surface Modification of AuNPs. Many amino acids can modify the surface of AuNPs through the Au−S bond, and they also have strong interactions with metal ions, such as Hg2+ or Pb2+. Based on the fact that Hg2+ coordinates with −NH2 and −COOH at C-terminus of 313

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Accounts of Chemical Research lysine and arginine, we modified AuNPs with arginine to prepare arginine-AuNPs (Arg-AuNPs), and Hg2+ can induce the aggregation of Arg-AuNPs. Because we can access several variables to produce the different outputs based on logic gate strategy, we designed versatile colorimetric logic gates depending on the specific coordination effects between amino acids38 (arginine, lysine, and cysteine) and metal ions (Hg2+ and Cr3+)(Figure 3A). Molecular logic gates39 can process chemical “inputs” to generate “outputs” based on the molecular interactions such as coordination-based recognition, which is potentially useful for biochemical assays. We set arginine and Hg2+ as two inputs, and set the color of AuNP solution as the output. For input, “0” represents the absence, while “1” represents the presence of arginine or Hg2+. For outputs, “0” represents red (dispersion) while “1” represents blue (aggregation) of the AuNPs. When both arginine and Hg2+ are present (two inputs are “1” and “1”), the AuNPs will be aggregated and show a blue color (the output is “1”); thus a colorimetric AND gate can be produced. However, conventional molecular logic gates performed in bulk often require expertise and consume plenty of reagents. To solve this problem, we further introduced microfluidic technology40 to demonstrate the molecular logic gates, and it proved to have merits of automated operation and visible readout of the logic gates (Figure 3B). The arginine-modified AuNPs can be also implemented for glutathione (GSH) detection based on the coordination recognition. We introduced a dispersion-dominated chromogenic strategy, which relies on the target-induced dispersion of AuNPs for detection of GSH based on Hg2+-mediated aggregation of Arg-AuNPs. In this method, Hg2+ can lead to the aggregation of Arg-AuNPs because of the molecular interactions between metal ions and amino acids. With the introduction of GSH, the specific interaction between GSH and Hg2+ prevented the aggregation of Arg-AuNPs, a biosensor can thus be developed for detection of GSH in biological samples41 (Figure 4). The LOD for GSH sensing is 10.9 × 10−9 M, and this method can be used to evaluate and quantify the GSH levels in cell lysates, revealing that cellular GSH level in cancer cells is higher than that in normal cells. However, few studies report the in vivo detection using AuNPs for naked-eye readout, considering that cell-level microscopic images based on AuNPs might not be available due to their nanometer particle size, as well as that it requires sufficient number of AuNPs to result in the visible color change. We also developed a colorimetric assay based on cysteinemediated aggregation of AuNPs42 (Figure 5A). Cysteine is an amino acid with a unique molecular structure: a thiol group (-SH) on one hand and NH2+ and COO− on the other hand. Cysteine can easily conjugate to the AuNPs through Au−S bond and “bridge” the AuNPs through the electrostatic interaction between positively and negatively charged groups that aggregate the AuNPs. Iodide can catalyze the oxidation of cysteine to form the disulfide cystine, and the latter could not cause the aggregation of AuNPs. Combining this catalytic process with HRP-catalyzed reaction, we developed a HRPmediated, iodide-catalyzed cascade reaction to modulate the dispersion/aggregation of AuNPs, which allowed for naked-eye readout. A separate protocol of surface modification of the AuNPs is not required for this immunoassay, making it easy to implement. This immunoassay has been used to analyze the hepatitis C virus-antibody in real serum samples with a detection accuracy of 100% by naked-eye, while it is 20% by

Figure 5. Amino acid-mediated surface modification of AuNPs for naked-eye analysis. (A) HRP-mediated modulation of the AuNPs for immunoassay. Iodide-catalyzed oxidation of cysteine can change the surface chemistry of AuNPs and regulate aggregation and dispersion of AuNPs. (B) Cd2+−GSH complex can change the surface property of AuNPs and improve the stability of the citrate−AuNPs when the concentration of NaCl changes.

conventional ELISA, demonstrating that this AuNP-based immunoassay allows for biomedical diagnostics with high sensitivity and naked-eye readout. The coordination between Cd2+ and GSH-AuNPs allows for detection of cadmium ions (Cd2+)43 (Figure 5B). The unmodified AuNPs easily aggregate in a high concentration of NaCl solution, but the presence of GSH can prevent the saltinduced aggregation of AuNPs. When Cd2+ is added to a stable mixture of AuNPs, GSH, and NaCl, one Cd2+ can coordinate with 4 × GSH, which decreases the amount of free GSH on the AuNPs to weaken the stability of AuNPs, and leads to their aggregation. This colorimetric method can successfully detect Cd2+ in water and digested rice samples with high accuracy and convenient operations. 2.2.2. Sulfhydryl Compound-Modified AuNPs. The chelation interaction between spiropyran (SP) and Cu2+ can be modulated by UV light, and SP can be isomerized to the planar and open merocyanine form (MC) after UV light irradiation, otherwise remaining in the nonplanar and closed SP form. We thus synthesized spiropyran-modified AuNPs (spiropyran-AuNPs) and utilized them to construct a resettable and multireadout logic system capable of several types of logic operations based on the aggregation of AuNPs in aqueous media44 (Figure 6A). Spiropyran-AuNPs are monodispersed in solutions to appear red in color. When exposed to UV, spiropyran turns into the open merocyanine, such that dispersed merocyanine-modified AuNPs (MC-AuNPs) will become aggregated with a color change from red to purple owing to the chelation between merocyanine and Cu2+. Importantly, the MC-AuNPs could easily return to the SP by 314

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Figure 6. Spiropyran-functionalized AuNPs realizes resettable, multireadout logic gates based on photoresponsive control. (A) Proposed mechanism for the photoresponsive control of spiropyran-AuNPs. (B) The spiropyran-AuNP-based resettable logic gates. (1) In the presence of both inputs (1/ 1), the color of the spiropyran-AuNP solutions changed from red to purple. (2) Photograph of AuNP solutions and (3) their corresponding UV/vis absorption. (4) A truth table of the AND logic gate. Adapted with permission from ref 44. Copyright 2011 Wiley.

Figure 7. Visual biosensors based on sulfhydryl compound-modified AuNPs for detection of heavy metal ions. (A) DMSA-functionalized AuNPs for rapid detection of both Cr(III) and Cr(VI). (B) The mixed charged thiols are used to modify the AuNPs to form intermolecular zwitterionic (Zw) surfaces. The M3+ including Fe3+, Al3+, and Cr3+ can effectively trigger the aggregation of Zw-AuNPs by interfering with their surface potential, and the aggregated AuNPs can be regenerated and recycled by removing M3+.

Detection of Cr3+ and Cr2O72− is very meaningful in environment monitoring. We have employed meso-2,3-

visible light, which means that the AuNP-based logic system is resettable for multiple times of detection (Figure 6B). 315

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Figure 8. Ligand exchange strategy for surface modification of AuNPs for biodetection. (A) Quaternary ammonium group-capped AuNPs for Hg2+ detection. (B) Rhodamine B isothiocyanate (RBITC)−poly(ethylene glycol) (PEG)-comodified AuNPs for Hg2 detection. (C) Rhodamine Bmodified AuNP-based assay for dual readout detection of acetylcholinesterase in cerebrospinal fluid of transgenic mice with Alzheimer’s disease. Adapted with permission from ref 49. Copyright 2012 Wiley. (D) Rhodamine B-AuNP-based assay with dual readouts for detection of organophosphorus and carbamate pesticides.

most previous reports on M3+ detection, the instrument-free and cost-saving features have made this assay ideal for use in resource-limited areas.

dimercaptosuccinic acid (DMSA) to modify AuNPs for detection of both Cr3+ and Cr2O72−45(Figure 7A). DMSA with two-SH groups and two -COOH groups was utilized as the reductant and stabilizer to synthesize Au NPs. The -COOH groups in the DMSA can be used to sense Cr3+. Meanwhile, one Cr2O7 2− interacts with two DMSA-AuNPs by strong OH···O hydrogen bonds through the end carboxyl groups of DMSA molecules. Density functional theory was used to calculate the change of the Gibbs free energy (ΔG) of the interactions between the DMSA-AuNPs and various metal ions. It shows that ΔG for Cr3+ and Cr2O72− is the lowest among all the metal ions, indicating that DMSA-AuNPs have highest specificity for both the hydrated form of Cr3+ and Cr2O72−. The LOD by the naked eye is 10 nM, which is much lower than the Environmental Protection Agency (EPA) standards (EPA3060A). This method has been successfully used to detect chromium ions in chromium-polluted soil from the chromium slag dumpsite. In addition to detection of specific ions, rapid and straightforward detection of trivalent metal ions (M3+) is important and challenging. We have synthesized zwitterionicmolecule-modified AuNPs (Zw-AuNPs). Aggregation of ZwAuNPs with a color change can be induced by M3+ including Fe3+, Al3+, and Cr3+ due to the intermolecular zwitterionic surface46 (Figure 7B). Furthermore, the Zw-AuNPs can be recycled because of the different status of trivalent ions in different pH values. Under acidic or neutral environments, M3+ can interact with Zw-AuNPs and induce their aggregation. When the solution becomes basic, free M3+ becomes M(OH)3 and inhibits the interaction between Zw-AuNPs and M3+, resulting in the redispersion of Zw-AuNPs. Compared with

2.3. Ligand Exchange Reaction of AuNPs for Biochemical Analysis

Ligand exchange for surface modification of AuNPs is an effective strategy to construct biosensors. We use several ligand exchange reactions to detect Hg2+ and a few important biochemical markers. We first adopted a quaternary ammonium group-terminated thiol (QA-SH) to modify the AuNPs and prepared the QA-AuNPs through Au−S bond47(Figure 8A). The QA-AuNPs are very stable in acidic solutions due to the electrostatic repulsion between the charged, quaternary ammonium groups of AuNPs. Because mercury(II) (Hg2+) has a stronger interaction with -SH group than QA-AuNPs, it could replace the QA-group from the AuNPs, which results in the aggregation of AuNPs due to the deprotection of thiolterminated QA. The degree of aggregation relates to the concentration of Hg2+; thus these QA-AuNPs can detect Hg2+ in aqueous solutions with a low LOD of 30 nM by the naked eye, much lower than the guideline value of drinking water set by the World Health Organization (WHO). Furthermore, we designed a recyclable probe by modifying rhodamine B isothiocyanate (RBITC) and poly(ethylene glycol) (PEG) on AuNPs for detection of Hg2+ with excellent robustness, selectivity, and sensitivity48(Figure 8B). Only Hg2+ can displace RBITC from the AuNP, resulting in the fluorescence enhancement of RBITC, which is initially quenched by AuNPs. Thiol-terminated PEG can bind with the remaining active sites of AuNPs to help AuNPs remain stable and monodisperse in real samples. The LOD of this assay 316

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Accounts of Chemical Research for Hg2+ (2.3 nM) was lower than the maximum limits guided by the EPA as well as that permitted by the WHO. The efficiency of this probe was demonstrated in monitoring Hg2+ in complex samples such as river water and living cells. This ligand exchange approach can detect thiols and other targets as well. rhodamine B-modified AuNPs (RB-AuNPs) are prepared to detect acetylcholinesterase (AChE), a biomarker for Alzheimer’s disease (AD). Rhodamine B can physically adsorb onto the AuNPs, and the AuNPs can quench the fluorescence of RB. When AChE is introduced, it can hydrolyze its substrate (acetylthiocholine, ATC) into thiocholine, which replaces RB from the AuNPs via the stronger Au−S bond compared to the physical adsorption between RB and AuNPs49(Figure 8C). Upon this ligand exchange reaction, the fluorescence of RB recovers, which can be used as for the quantitative detection of AChE. Meanwhile, the dispersed AuNPs become aggregated via electrostatic interaction between the positively charged thiocholine and the negatively charged citrate from the AuNPs. The state change of AuNPs (from dispersed to aggregated) results in the color change of AuNPs (from red to blue), which can be used as visual signal for the detection of AChE. The dual readouts, fluorometric and colorimetric, provided a sensitive and selective assay for detecting at least 0.1 mU/mL of AChE in the cerebrospinal fluid (CSF) of transgenic mice suffering from AD. This RBAuNP-based assay could thus be promising for monitoring AChE in human CSF for early diagnosis and prognosis of AD. The RB-AuNPs can detect organophosphorus and carbamate pesticides in food samples because these two common organophosphorus pesticides can effectively inhibit the activity of AChE50 (Figure 8D). If the food samples are polluted by these organophosphorus pesticides, the AChE cannot hydrolyze the ATC into thiocholine, and thiocholine will lead to the aggregation of AuNPs through electrostatic interactions. The readout of this assay is thus the reverse of the assay for AChE, as higher fluorescence and blue color indicate the negative sample. The LOD by both the naked eye and fluorescence signal was enough to satisfy the maximum residue limits (MRL) as required by the EPA. Compared with conventional methods, the biggest advantage of this strategy is that this dual readout system that can meet different needs, where fluorescence signal can be used as the quantitative signal in large testing laboratory and the color of AuNPs can be used as the qualitative signal to realize on-site testing.

complex biological samples. Computer simulation can provide useful information for optimizing performance in the development of nanoparticle-based biosensors, which can greatly improve the accuracy and reduce the cost. (2) To manufacture useful products, robust, low-cost standard protocols for reproducible, mass scale production of stable functionalized AuNPs are required. Compared with conventional synthetic methods, microfluidic systems will likely have outstanding advantages in the extremely low reaction volumes, rapid throughput, and real-time tuning of product properties in continuousflow formats. (3) New nanomaterials and new types of reactions (new organic/inorganic reactions) will help develop new types of analytical tools, which can provide more choices for surface modification of NPs and may improve the performance of biochemical assays.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Xingyu Jiang: 0000-0002-5008-4703 Author Contributions #

Y.C. and Y.X. equally contributed to this work.

Notes

The authors declare no competing financial interest. Biographies Yiping Chen obtained his B.S. and M.S. at Huazhong Agricultural University (2003−2007), followed by a Ph.D. (2013) from Beijing Normal University, working on magnetic nanoparticle-mediated biochemical analysis. He joined the NCNST as an assistant professor in 2013 and got promoted to be an associate professor in 2016. Yiping’s research interests focus on nanoparticle-mediated immunosensors for point-of-care diagnostics, environmental monitoring, and food safety. Yunlei Xianyu obtained his B.S. at Huazhong University of Science and Technology (2011), followed by a Ph.D. (2016) from University of Chinese Academy of Sciences, working on gold nanparticles and biosensors with Prof. Xingyu Jiang. He is currently a Research Associate in the Department of Materials at Imperial College London.

3. DISCUSSION AND OUTLOOK AuNP-based biochemical analysis has the potential to enhance or supersede current analytical techniques. However, AuNPmediated assays still have many limitations (sensitivity, detection efficiency, and stability) that should be addressed to ensure they have unparalleled advantages to replace conventional analytical methods. Future success depends on the continuation of such research endeavors, and we anticipate that the research interests and attention should be focused on taking these AuNP-based assays from being lab curiosities to achieving high-impact practical applications. To realize this goal, the following measures are necessary: (1) A series of surface chemistry strategies should be developed, including the capping agents and bioconjugation method. Effective capping agents yield high stability and strong anti-interference ability to improve the quality of AuNPs, which is essential for their use in

Xingyu Jiang obtained his B.S. at the University of Chicago (1999), followed by an A.M. (2001) and a Ph.D. (2004) from Harvard University (Chemistry), working on microfluidics and cell patterning with Prof. George Whitesides. He joined the NCNST in 2005 where he has remained since. Xingyu’s research interests include gold nanparticles, surface chemistry, microfluidics, cell biology, immunoassays, and nanomedicine.

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ACKNOWLEDGMENTS We thank Y. Cheng for helping with the TOC graphic. We also thank the Ministry of Science and Technology of China (Grant 2013YQ190467) and the National Science Foundation of China (Grants 21505027, 21535001, 51373043, and 81361140345). 317

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