Redox-active ionic-liquid-assisted one-step general method for preparing gold nanoparticle thin films: applications in refractive index sensing and catalysis

We describe a general one-step facile method for depositing gold nanoparticle (GNP) thin films onto any type of substrates by the in situ reduction of AuCl(3) using a newly designed redox-active ionic liquid (IL), tetrabutylphosphonium citrate ([TBP][Ci]). Various substrates such as positively charged glass, negatively charged glass/quartz, neutral hydrophobic glass, polypropylene, polystyrene, plain paper, and cellophane paper are successfully coated with a thin film of GNPs. This IL ([TBP][Ci]) is prepared by the simple neutralization of tetrabutylphosphonium hydroxide with citric acid. We also demonstrate that the [TBP][Ci] ionic liquid can be successfully used to generate GNPs in an aqueous colloidal suspension in situ. The deposited GNP thin films on various surfaces are made up of mostly discrete spherical GNPs that are well distributed throughout the film, as confirmed by field-emission scanning electron microscopy. However, it seems that some GNPs are arranged to form arrays depending on the nature of surface. We also characterize these GNP thin films via UV-vis spectroscopy and X-ray diffractometry. The as-formed GNP thin films show excellent stability toward solvent washing. We demonstrate that the thin film of GNPs on a glass/quartz surface can be successfully used as a refractive index (RI) sensor for different polar and nonpolar organic solvents. The as-formed GNP thin films on different surfaces show excellent catalytic activity in the borohydride reduction of p-nitrophenol.     

One-Step Synthesis of Stable Colloidal Gold Nanoparticles Through Bioconjugation with Bovine Serum Albumin in Harsh Environments

In saline solutions with NaCl concentrations less than that typical of blood plasma and bodily fluids, gold nanoparticles (GNPs) aggregate and precipitate because of GNP cation complexation with the Cl^? anions in the solution. It is difficult to retain stable colloidal GNPs within any saline solution for a relatively long time without aggregation and precipitation. In this study, we developed a method to synthesize stable GNPs in harsh anion-containing environments. GNPs were formed by laser ablation in a saline solution, and their stabilization was achieved by adding bovine serum albumin (BSA) to the NaCl solution; this has been shown to be a quick, efficient approach to producing stable colloidal GNPs. GNP nanoclusters in saline solutions with and without BSA were observed via high-resolution transmission electron microscopy and selected area electron diffraction. The results reveal that our methodology yields colloidal GNPs with long-term stability in a BSA-containing saline solution.     

Effect of size and protein environment on electrochemical properties of gold nanoparticles on carbon electrodes

We studied the electrochemical properties of gold nanoparticles (GNPs) and their complexes with proteins using square-wave voltammetry. Effect of the nanoparticle size and detection procedure was explored upon the oxidation of GNPs on a glassy carbon electrode (GCE). For pre-characterized GNPs of 13, 35 and 78 nm diameter, the oxidation peak potential was + 0.98, + 1.03 and + 1.06 V vs. Ag/AgCl, respectively. The conjugation of GNPs with four different proteins was verified by UV–Vis spectroscopy and atomic force microscopy indicated the formation of protein shells around GNPs. This process hampered the oxidation of GNPs on bare GCE causing pronounced decrease in the current response by an average factor of 72. GCE modification with carbon nanotubes weakly influenced the sensitivity of GNP detection but resulted in a 14.5-fold signal increase averaged for all GNP–protein complexes. The acidic dissolution and electrodeposition of GNPs or their complexes adsorbed on GCE allowed superior signal amplification directly proportional to nanoparticle size. The results are useful for the optimization of voltammetric analysis of GNP–protein complexes and can be extended to the characterization of other metal nanostructures and their complexes with biological components.     

Correlation between catalytic activity and surface ligands of monolayer protected gold nanoparticles

Gold nanoparticles (GNPs) are known to be a very good catalyst. Also, the anchoring of GNPs with stabilizing ligands is essential for surface modification, tuning of size and shapes, and to prevent from aggregation in suspension. But the effect of ligand on the catalytic property of ligand-capped GNP is yet to be explored in detail. In this paper, we perform an in-depth study of effect of ligands on the catalytic activity of monolayer protected GNPs. For this study, a series of different ligand functionalized GNPs in suspension as well as functionalized GNPs' thin film on glass substrate are prepared and used as catalysts in two model reactions, viz. borohydride reduction of 4-nitrophenol and redox reaction between potassium ferricyanide and sodium thiosulfate. The functionalization of GNPs with any ligand reduces its virgin catalytic activity, no matter whether the GNPs are suspended or supported as thin film. An increase in alkyl chain length of alkanethiols and alkylamines ligands and their graft density to the surface of GNP reduces its catalytic activity. Interestingly, the capping of GNPs with 11-mercaptoundecanoic acid and 11-mercaptoundecanol ligands completely destroys its catalytic activity. The effect of anchoring group of ligand molecules on the catalytic activity of ligand-protected GNPs is also discussed.     

Disclosure of the imidazolium cation coordination and stabilization mode in ionic liquid stabilized gold(0) nanoparticles

A surface-enhanced Raman spectroscopy (SERS) study of imidazolium ionic liquid stabilized gold(0) nanoparticles (GNPs) furnished previously unknown knowledge about the coordination and stabilization mode of the imidazolium cation. GNPs were prepared by hydrazine reduction of a chloroauric acid solution in 1-triethylene glycol monomethyl ether-3-methylimidazolium methanesulfonate 2 as ether-functionalized room-temperature ionic liquid (RTIL). UV-vis spectroscopy showed the presence of GNP aggregates as absorptions extended to the NIR region. A parallel coordination mode for the imidazolium cation of RTIL 2 on the GNP surface was observed by SERS, which occurred without the simultaneous coordination of the 1-triethylene glycol monomethyl ether-functionality. Instead of this, the ether-functionality was directed away from the GNP surface and acted as steric barrier between the GNPs/GNP aggregates, thus preventing further aggregation. These new insights suggest that the imidazolium cation is responsible for electrosteric stabilization.     

Fractionation and characterization of gold nanoparticles in aqueous solution: asymmetric-flow field flow fractionation with MALS, DLS, and UV–Vis detection

Asymmetrical-flow field flow fractionation (AFFF) separates constituents based on hydrodynamic size and is emerging as a powerful tool for obtaining high-resolution information on the size, molecular weight, composition, and stability of nanoscale particles in liquid media. We employ a customized AFFF system combining on-line detectors for multi-angle light scattering, dynamic light scattering, and UV–Vis absorption. Our objective is to develop optimized measurement protocols for the characterization of gold nanoparticles (GNPs), which are widely utilized in biomedical research and other nanotechnology applications. Experimental conditions have been optimized by controlling key parameters, including injection volume and solids concentration, mobile phase composition, membrane type and pore size, and ratio of channel-to-cross-flow rates. Individual citrate-stabilized GNP components (nominally 10, 20, 30, 40, and 60 nm) and GNPs functionalized with polyethylene glycol were separated from multicomponent GNP mixtures by AFFF and subsequently characterized. We discuss the effects due to variations in measurement parameters and GNP surface modification on observed retention, recovery, and peak resolution.     

Gold nanoparticles enhance the X-ray-induced degradation of human centrin 2 protein

In the war against cancer, radiotherapy is a prominent tool but counterbalanced by the fact that it also induces damages in healthy tissues. Nanotechnologies could open a new possibility to decrease these side effects. In particular, gold nanoparticles (GNPs) could be used as radio-sensitizers. As the role of proteins in the processes leading to cell death cannot be neglected, their radio-sensitization by GNPs is of great interest. This is particularly true in the case of the human centrin 2 protein, which has been proposed to be involved in DNA repair processes. To investigate this effect, we quantified for the first time the degradation of this protein in a gold colloidal solution when submitted to X-rays. We showed that the X-ray-induced degradation of the human centrin 2 protein is enhanced 1.5-fold in the presence of GNPs, even though no covalent bond exists between protein and GNPs. Among the conditions tested, the maximum enhancement was found with the higher GNP:protein ratio of 2×10⁻⁴ and with the higher X-ray energy of 49 keV.     

Completely dispersible PEGylated gold nanoparticles under physiological conditions: modification of gold nanoparticles with precisely controlled PEG-b-polyamine

A novel water-soluble, biocompatible polymer, poly(ethylene glycol)-block-poly((2-N,N-dimethylamino)ethyl methacrylate) (PEG-b-PAMA), possessing controlled molecular weight with a narrow molecular weight distribution, was synthesized by the atom-transfer radical polymerization (ATRP) method. PEG-b-PAMA having a short PAMA chain length was successfully synthesized under suitable polymerization conditions. Gold nanoparticles (GNPs) were modified using PEG-b-PAMA prepared under a variety of PEGylation conditions. Under alkaline conditions (pH >10) and an [N]/[GNP] ratio of more than 3300, the PEGylated GNPs (PEG-GNPs) showed complete dispersion stability, avoiding coagulation. The amino groups of the PAMA segment of the block copolymers were completely deprotonated above pH 10. This means that PEG-b-PAMA interacted with the GNP surface via multipoint coordination of the tertiary amino groups of PAMA, not electrostatically. The effect of the number of amino groups in the PAMA segment on GNP surface modifications was investigated by zeta potential and dynamic light scattering (DLS) measurements. When the PEG-GNPs were prepared in excess polymer solution, almost the same diameter was observed regardless of the PAMA chain length. After the PEG-GNPs were purified by centrifugation, the zeta potentials of all PEG-GNPs were shielded to almost 0 mV, indicating the effective modifications of the GNP surface by PEG-b-PAMA regardless of the chain length. However, the particle size and particle size distribution of the purified PEG-GNPs were strongly affected by the PAMA chain length. PEG-GNPs with longer PAMA segments underwent coagulation after purification, whereas PEG-GNPs with shorter PAMA segments increased their dispersion stability. The experimental results of the thermal gravimetric analysis confirmed that the PEG density on the GNP surface increased as the AMA units decreased to 3. Thus, the dispersion stability depended significantly on the PEG density on the GNP surface. GNPs modified with PEG-b-PAMA having short AMA units showed excellent dispersion stability under a variety of pH conditions. The excellent dispersion stability of the obtained PEG-GNP was also confirmed both in bovine serum albumin (BSA) solution and 95% human serum.     

Gold nanoparticle–antibody conjugates for specific extraction and subsequent analysis by liquid chromatography–tandem mass spectrometry of malondialdehyde-modified low density lipoprotein as biomarker for cardiovascular risk

Oxidized low-density lipoproteins (OxLDLs) like malondialdehyde-modified low-density lipoprotein (MDA-LDL) play a major role in atherosclerosis and have been proposed as useful biomarkers for oxidative stress. In this study, gold-nanoparticles (GNPs) were functionalized via distinct chemistries with anti-MDA-LDL antibodies (Abs) for selective recognition and capture of MDA-LDL from biological matrices. The study focused on optimization of binding affinities and saturation capacities of the antiMDA-LDL-Ab-GNP bioconjugate by exploring distinct random and oriented immobilization approaches, such as (i) direct adsorptive attachment of Abs on the GNP surface, (ii) covalent bonding by amide coupling of Abs to carboxy-terminated-pegylated GNPs, (iii) oriented immobilization via oxidized carbohydrate moiety of the Ab on hydrazide-derivatized GNPs and (iv) cysteine-tagged protein A (cProtA)-bonded GNPs. Depending on immobilization chemistry, up to 3 antibodies per GNP could be immobilized as determined by ELISA. The highest binding capacity was achieved with the GNP-cProtA-Ab bioconjugate which yielded a saturation capacity of 2.24 ± 0.04 μg mL⁻¹ GNP suspension for MDA-LDL with an affinity K_d of 5.25 ± 0.11 × 10⁻¹⁰ M. The GNP-cProtA-antiMDA-LDL bioconjugate revealed high specificity for MDA-LDL over copper(II)-oxidized LDL as well as native human LDL. This clearly demonstrates the usefulness of the new GNP-Ab bioconjugates for specific extraction of MDA-LDL from plasma samples as biomarkers of oxidative stress. Their combination as specific immunoextraction nanomaterials with analysis by LC–MS/MS allows sensitive and selective detection of MDA-LDL in complex samples.     

Functionalized gold nanoparticles for sample preparation: A review

Sample preparation is a crucial step for the reliable and accurate analysis of both small molecule and biopolymers which often involves processes such as isolation, pre‐concentration, removal of interferences (purification), and pre‐processing (e.g., enzymatic digestion) of targets from a complex matrix. Gold nanoparticle (GNP)‐assisted sample preparation and pre‐concentration has been extensively applied in many analytical procedures in recent years due to the favorable and unique properties of GNPs such as size‐controlled synthesis, large surface‐to‐volume ratio, surface inertness, straightforward surface modification, easy separation requiring minimal manipulation of samples. This review article primarily focuses on applications of GNPs in sample preparation, in particular for bioaffinity capture and biocatalysis. In addition, their most common synthesis, surface modification and characterization methods are briefly summarized. Proper surface modification for GNPs designed in accordance to their target application directly influence their functionalities, e.g., extraction efficiencies, and catalytic efficiencies. Characterization of GNPs after synthesis and modification is worthwhile for monitoring and controlling the fabrication process to ensure proper quality and functionality. Parameters such as morphology, colloidal stability, and physical/chemical properties can be assessed by methods such as surface plasmon resonance, dynamic light scattering, ζ‐potential determinations, transmission electron microscopy, Taylor dispersion analysis, and resonant mass measurement, among others. The accurate determination of the surface coverage appears to be also mandatory for the quality control of functionality of the nanoparticles. Some promising applications of (functionalized) GNPs for bioanalysis and sample preparation are described herein.     

Cellular Uptake of Gold Nanoparticles and Their Movement in 3D Multicellular Tumor Spheroids: Effect of Molecular Weight and Grafting Density of Poly(2‐hydroxyl ethyl acrylate)

It is known that the size of gold nanoparticles (GNPs) is not the only determining factor in the uptake by cells such as cancer cells. The surface functionalization plays a crucial role, in particular the nature of the ligand as well as the molecular weight and the grafting density. Here, poly(2‐hydroxy ethyl) acrylate (pHEA) with molecular weights ranging from 10, 20 to 39 g mol^?1 via reversible addition–fragmentation chain transfer polymerization is synthesized. These polymers are used directly to coat GNPs with sizes of 20, 40, and 70 nm as the trithiocarbonate functionality can strongly bind to the gold surface. The library of nine GNP is found to be nontoxic against lung carcinoma cells A549 and has negligible albumin protein absorption as determined by quartz crystal microbalance. Laser scanning confocal microscopy and flow cytometry reveal that GNP coated with medium length pHEA displays the highest cellular uptake while the effect of the size is not statistically significant. In contrast, multicellular tumor spheroids, which is a 3D model that simulates the tissue, enable the penetration of GNP coated with the longest pHEA chain while it also appears that smaller GNPs have now a clear advantage.     

Influence of Gold Nanoparticles of Varying Size in Improving the Lipase Activity within Cationic Reverse Micelles

Herein, we report the effect of gold nanoparticles (GNPs) in enhancing lipase activity in reverse micelles of cetyltrimethylammonium bromide (CTAB)/water/isooctane/n‐hexanol. The size and concentration of the nanoparticles were varied and their specific roles were assessed in detail. An overall enhancement of activity was observed in the GNP‐doped CTAB reverse micelles. The improvement in activity becomes more prominent with increasing concentration and size of the GNPs (0–52 μM and ca. 3–30 nm, respectively). The observed highest lipase activity (k₂=1070±12 cm³ g^?1 s^?1) in GNP‐doped CTAB reverse micelles ([GNP]: 52 μm, ca. 20 nm) is 2.5‐fold higher than in CTAB reverse micelles without GNPs. Improvement in the lipase activity is only specific to the GNP‐doped reverse micellar media, whereas GNP deactivates and structurally deforms the enzyme in aqueous media. The reason for this activation is probably due to the formation of larger‐sized reverse micelles in which the GNP acts as a polar core and the surfactants aggregate around the nanoparticle (‘GNP pool’) instead of only water. Lipase at the augmented interface of the GNP‐doped reverse micelle showed improved activity because of enhancement in both the substrate and enzyme concentrations and increased flexibility in the lipase conformation. The extent of the activation is greater in the case of the larger‐sized GNPs. A correlation has been established between the activity of lipase and its secondary structure by using circular dichroism and FTIR spectroscopic analysis. The generalized influence of GNP is verified in the reverse micelles of another surfactant, namely, cetyltripropylammonium bromide (CTPAB). TEM, dynamic light scattering (DLS), and UV/Vis spectroscopic analysis were utilized to characterize the GNPs and the organized aggregates. For the first time, CTAB‐based reverse micelles have been found to be an excellent host for lipase simply by doping with appropriately sized GNPs.     

Striking Improvement in Peroxidase Activity of Cytochrome c by Modulating Hydrophobicity of Surface‐Functionalized Gold Nanoparticles within Cationic Reverse Micelles

This work demonstrates a remarkable enhancement in the peroxidase activity of mitochondrial membrane protein cytochrome c (cyt c) by perturbing its tertiary structure in the presence of surface‐functionalised gold nanoparticles (GNPs) within cetyltrimethylammonium bromide (CTAB) reverse micelles. The loss in the tertiary structure of cyt c exposes its heme moiety (which is buried inside in the native globular form), which provides greater substrate (pyrogallol and H₂O₂) accessibility to the reactive heme residue. The surfactant shell of the CTAB reverse micelle in the presence of co‐surfactant (n‐hexanol) exerted higher crowding effects on the interfacially bound cyt c than similar anionic systems. The congested interface led to protein unfolding, which resulted in a 56‐fold higher peroxidase activity of cyt c than that in water. Further perturbation in the protein’s structure was achieved by doping amphiphile‐capped GNPs with varying hydrophobicities in the water pool of the reverse micelles. The hydrophobic moiety on the surface of the GNPs was directed towards the interfacial region, which induced major steric strain at the interface. Consequently, interaction of the protein with the hydrophobic domain of the amphiphile further disrupted its tertiary structure, which led to better opening up of the heme residue and, thereby, superior activity of the cyt c. The cyt c activity in the reverse micelles proportionately enhanced with an increase in the hydrophobicity of the GNP‐capping amphiphiles. A rigid cholesterol moiety as the hydrophobic end group of the GNP strikingly improved the cyt c activity by up to 200‐fold relative to that found in aqueous buffer. Fluorescence studies with both a tryptophan residue (Trp59) of the native protein and the sodium salt of fluorescein delineated the crucial role of the hydrophobicity of the GNP‐capping amphiphiles in improving the peroxidase activity of cyt c by unfolding its tertiary structure within the reverse micelles.     

Synthesis and capping of water-dispersed gold nanoparticles by an amino acid: bioconjugation and binding studies

We report a novel strategy for the synthesis of aqueous stable, carboxylated gold nanoparticles (GNPs) by using glutamic acid as the reducing agent. The ratio of chloroaurate ions, AuCl(-)(4) to glutamic acid was optimized in the reaction medium to obtain monodispersed GNPs. Glutamic acid reduced gold nanoparticles were characterized by UV-visible, FTIR, dynamic light scattering and transmission electron microscopy, which demonstrated high stability in aqueous solution over a period of time indicating stabilization via surface-bound amino acid. Functionalized nanoparticles were conjugated with protein molecules through electrostatic attraction between the surface-terminated negatively charged carboxylate groups (COO(-)) of glutamic acid and the positively charged amino groups (NH(+)(3)) of the protein. The conjugation efficiency of the GNP:protein conjugates was confirmed qualitatively and quantitatively through gel electrophoresis and critical flocculation concentration analysis. The interaction between functionalized GNPs with protein molecules was investigated using fluorescence spectroscopy showing the fluorescence quenching of the tryptophan residues of protein molecules after conjugation. Circular dichroism (CD) studies of the conjugates confirmed that the protein undergoes a more flexible conformational state on the boundary surface of GNPs after conjugation. There was substantial conformational transition from alpha-helix to beta-sheet structure after conjugation of protein to GNPs.     

纳米金与牛血清白蛋白作用的毛细管电泳研究

开展了针对微量纳米金与牛血清白蛋白相互作用的毛细管电泳研究, 测得二者的结合常数为28.6 L/μmol, 每个纳米金颗粒吸附约24个牛血清白蛋白分子. 结果表明, 牛血清白蛋白可改善并稳定纳米金的峰形, 二者作用时温育介质的pH以及电泳所用的缓冲溶液浓度对毛细管电泳(CE)效率有重要影响. 此法可推广到其它纳米颗粒的吸附研究中.     

Effect of surface oxidation on the interaction of 1-methylaminopyrene with gold nanoparticles

The effect of the surface chemistry of gold nanoparticles (GNPs) on the GNP-amine (-NH(2)) interaction was investigated via conjugating an amine probe--1-methylaminopyrene (MAP) chromophore--with three Au colloidal samples of the same particle size yet different surface chemistry. The surface of laser-irradiated and ligand-exchanged-irradiated GNPs is covered with acetonedicarboxylic ligands (due to laser-introduced citrate oxidization) and citrate ligands, respectively, and both surfaces contain oxidized Au species which are essentially lacking for the citrate-capped GNPs prepared by the pure chemical approach. Both laser-irradiated samples show inferior adsorption capacity of MAP as compared with the purely chemically prepared GNPs. Detailed investigations indicate that MAP molecules mainly complex directly with Au atoms via forming Au-NH(2)R bonds, and the oxidization of the GNP surface strongly influences the ratio of this direct bonding to the indirect bonding originating from the electrostatic interaction between protonated amine (-NH(3)(+)) and negatively charged surface ligands. The impact of the oxidized GNP surface associated with the laser treatment is further confirmed by aging experiment on GNP-MAP conjugation systems, which straightforwardly verifies that the surface oxidation leads to the decrease in the MAP adsorption on GNPs.     

Effects of novel quasi-interpenetrating network/gold nanoparticles composite matrices on DNA sequencing performances by CE

Gold nanoparticles (GNPs) with particle sizes of about 20, 40, and 60 nm were prepared and added into a quasi-interpenetrating network (quasi-IPN) composed of linear polyacrylamide (LPA) with different viscosity-average molecular masses of 1.5, 3.3, and 6.5 MDa and poly-N,N-dimethylacrylamide (PDMA) to form polymer/metal composite matrices, respectively. These novel matrices could improve ssDNA sequencing performances due to interactions between GNPs and polymer chains and the formation of physical cross-linking points as demonstrated by intrinsic viscosities and glass transition temperatures. The effects of the parameters in relation to quasi-IPN/GNPs matrices, such as GNP contents, GNP particle sizes, LPA molecular masses, and solution concentrations, on ssDNA sequencing performances were studied. In the presence of GNPs, the separation had the advantages of high resolution, speediness, excellent reproducibility, long shelf life and easy automation. Therefore, less viscous matrix solutions (with moderate size GNPs) due to lower solution concentration and lower-molecular-mass LPA could be used to replace more viscous solutions (without GNPs) due to higher solution concentration or higher-molecular-mass LPA to separate DNA, while the sieving performances were approximate even higher, which helped to achieve full automation especial for capillary array electrophoresis (CAE) and microchip electrophoresis (MCE).     

Graphene Nanoplatelets: Electrochemical Properties and Applications for Oxidation of Endocrine‐Disrupting Chemicals

In most graphene‐based electrochemical applications, graphene nanoplatelets (GNPs) have been applied. Now, for the first time, electrochemical properties of GNPs, namely, its electrochemical activity, potential window, and double‐layer capacitance, have been investigated. These properties are compared with those of carbon nanotubes (CNTs). GNP‐ and CNT‐coated electrodes were then applied for electrochemical oxidation of endocrine‐disrupting chemicals. The GNP‐coated electrode was characterized by atomic force microscopy and electrochemical techniques. Compared with the CNT‐coated electrode, higher peak current for the oxidation of 4‐nonylphenol is achieved on the GNP‐coated electrode, together with lower capacitive current. Electrochemical oxidation of 2,4‐dichlorophenol, bisphenol A, and octylphenol in the absence or presence of 4‐nonylphenol was studied on the GNP‐coated electrode. The results suggest that GNPs have better electrochemical performance than CNTs and are thus more promising for electrochemical applications, for example, electrochemical detection and removal of endocrine‐disrupting chemicals.     

Gold nanoparticle-fluorophore complex for conditionally fluorescing signal mediator

Fluorescent contrast agents with high specificity and sensitivity are valuable for accurate disease detection and diagnosis. Spherical gold nanoparticles (GNPs) can be smartly utilized for developing highly effective agents. The strong electromagnetic (plasmon) field on their surface can be very effective in influencing the electrons of fluorophores and, thus, manipulating the fluorescence output (i.e., either quenching or enhancement). Fluorescence quenching can be used for negative sensing, or for conditional de-quenching to increase the specificity. Fluorescence enhancement allows sensing to be more sensitive. The level of fluorescence alteration depends on the GNP size, the excitation and emission wavelengths and quantum yield of the fluorophore, and the distance between the GNP and the fluorophore. To understand the mechanisms of the fluorescence change by GNP, we have theoretically analyzed the parameters involved in the fluorescence alteration for commonly used fluorophores, with an emphasis on quenching. The results showed that the fluorescence of fluorophores with the excitation (Ex) and emission (Ex) wavelengths close to the GNP resonance peak tended to be significantly quenched by GNPs. For those fluorophores emitting fluorescence in red or near infrared, to achieve quenching, the distance between GNP and the fluorophore was required to be very short. In general, a shorter distance resulted in more quenching. Bigger GNPs require a shorter distance to achieve the same level of quenching. The fluorescence of a fluorophore with a lower quantum yield (especially the one with emission in far-red or near-infrared) is more difficult to be quenched by GNPs (requires very short distance). Instead, it can be enhanced. Based on the theoretical study, we have developed a near-infrared contrast agent, i.e., Cypate conjugated GNP via a short peptide spacer. Normally the fluorescence of Cypate was quenched. The spacer has a motif of a substrate for urokinase type plasminogen activator (uPA; cancer-secreting enzyme). This contrast agent emits fluorescence only in the presence of uPA, where the uPA cleaves the spacer. This design can be used in characterization of the cancer type and also in diagnosing other diseases with signature enzymes.