Silver and gold glyconanoparticles for colorimetric bioassays

The color changes associated with the aggregation of metal nanoparticles has led to the development of colorimetric-based assays for a variety of target species. We have examined both silver- and gold-based nanoparticles in order to establish whether either metal exhibits optimal characteristics for bioassay development. These silver and gold nanoparticles have been stabilized with a self-assembled monolayer of a mannose derivative (2-mercaptoethyl alpha-d-mannopyranoside) with the aim of inducing aggregation by exploiting the well-known interaction between mannose and the lectin Concanavalin A (Con A). Both metal glyconanoparticles were determined to be ca. 16 nm in diameter (using TEM measurements). Aggregation was observed on addition of Con A to both silver and gold nanoparticles resulting in a shift in the surface plasmon absorption band and a consequent color change of the solution, which was monitored using UV-visible spectrophotometry. Mannose-stabilized silver nanoparticles at a concentration of 3 nM provide an assay for Con A with the largest linear range (between 0.08 and 0.26 microM). Additionally, the kinetic rate of aggregation of the silver-nanoparticle-based bioassay was significantly greater than that of the gold-nanoparticle system. However, in terms of sensitivity, the mannose-stabilized gold-nanoparticle-based assay was optimum with a limit of detection of 0.04 microM Con A, as compared with a value of 0.1 microM obtained for the mannose-stabilized silver nanoparticles. Additionally, a lactose derivative (11-mercapto-3,6,9-trioxaundecyl beta-D-lactoside) was used to stabilize gold nanoparticles to induce aggregation upon addition of the galactose specific lectin Ricinus communis agglutinin (RCA(120)). To examine the specificity of the bioassay, lactose-stabilized gold nanoparticles were mixed with a solution of mannose-stabilized silver nanoparticles to give an aggregation assay capable of detecting two different lectins. When either Con A or RCA(120) was added to the mixed glyconanoparticles, selective recognition of the respective natural ligand was shown by aggregation of a single metal nanoparticle. Centrifugation and removal of the aggregated species enabled further bioassay measurements using the second glyconanoparticle system.     

Quantitative and reversible lectin-induced association of gold nanoparticles modified with alpha-lactosyl-omega-mercapto-poly(ethylene glycol)

Gold nanoparticles (1-10 nm size range) were prepared with an appreciably narrow size distribution by in situ reduction of HAuCl(4) in the presence of heterobifunctional poly(ethylene glycol) (PEG) derivatives containing both mercapto and acetal groups (alpha-acetal-omega-mercapto-PEG). The alpha-acetal-PEG layers formed on gold nanoparticles impart appreciable stability to the nanoparticles in aqueous solutions with elevated ionic strength and also in serum-containing medium. The PEG acetal terminal group was converted to aldehyde by gentle acid treatment, followed by the reaction with p-aminophenyl-beta-D- lactopyranoside (Lac) in the presence of (CH(3))(2)NHBH(3). Lac-conjugated gold nanoparticles exhibited selective aggregation when exposed to Recinus communis agglutinin (RCA(120)), a bivalent lectin specifically recognizing the beta-D-galactose residue, inducing significant changes in the absorption spectrum with concomitant visible color change from pinkish-red to purple. Aggregation of the Lac-functionalized gold nanoparticles by the RCA(120) lectin was reversible, recovering the original dispersed phase and color by addition of excess galactose. Further, the degree of aggregation was proportional to lectin concentration, allowing the system to be utilized to quantitate lectin concentration with nearly the same sensitivity as ELISA. This simple, yet highly effective, derivatization of gold nanoparticles with heterobifunctional PEG provides a convenient method to construct various colloidal sensor systems currently applied in bioassays and biorecognition.     

Fluorescence Turn‐On Sensing of Lectins with Mannose‐Substituted Tetraphenylethenes Based on Aggregation‐Induced Emission

The synthesis of mannose‐substituted tetraphenylethenes (TPEs) and their aggregation‐induced emission (AIE) behavior, induced by interactions with concanavalin A (Con A), are reported. A mixture of the mannose‐TPE conjugates and Con A in a buffer solution displays an intense blue emission on agglutination within a few seconds, which serves as a “turn‐on” fluorescent sensor for lectins. The sensing is also selective: the conjugates act as a sensor for Con A, but do not sense a galactose‐binding lectin, PNA. Con A‐recognition is not affected even in the presence of other proteins in a mixture. The conjugates also exhibit high sensitivity to detect Con A. An increased sensitivity of the conjugates results if mannopyranoside substituents are linked to the TPE‐core unit with a flexible chain and/or when the number of mannose residues increases.     

Binding of Ricinus communis agglutinin to a galactose-carrying polymer brush on a colloidal gold monolayer

A polymer with many pendent galactose residues was prepared by atom-transfer radical polymerization (ATRP) of galactose-carrying vinyl monomer, 2-lactobionamidoethyl methacrylate (LAMA), with a disulfide-carrying ATRP initiator, 2-(2'-bromoisobutyroyl)ethyl disulfide (DT-Br). The galactose-carrying polymer obtained (DT-PLAMA) was accumulated as a polymer brush via Au-S bond on a colloidal gold monolayer deposited on a cover glass. For comparison, a disulfide which carried one galactose residue at both ends (2-lactobionamidoethyl disulfide, Cys-Lac) was accumulated as a self-assembled monolayer (SAM) on the colloidal gold monolayer, too. The association and dissociation processes of galactose residues on the colloidal gold with a lectin, Ricinus communis agglutinin (RCA(120)), were observed by the increase and decrease in absorbance at 550nm corresponding to localized surface plasmon resonance (LSPR) phenomena. The Cys-Lac SAM-carrying glass chip showed a strong non-specific adsorption of the lectin, whereas the DT-PLAMA brush-carrying one reversibly associated with the lectin, indicating reusability of the latter device. The apparent association constant of the lectin with the galactose residues in the DT-PLAMA brush was much larger than the association constant for free galactose, and the detection limit of RCA(120) by the glycopolymer brush-modified device was satisfactorily low. Furthermore, a microscopic observation clearly indicated that the DT-PLAMA brush could reversibly associate with a HepG2 cell having galactose receptors, though these processes could not be observed spectrophotometrically due to a gigantic size of the cell.     

12-Mercaptododecyl β-maltoside-modified gold nanoparticles: specific ligands for concanavalin A having long flexible hydrocarbon chains

A simple and highly specific protein detection system using glycoconjugated gold nanoparticles was investigated. This system was based on the aggregation of gold nanoparticles coated with carbohydrate alkanethiols in the presence of corresponding proteins (lectins) that had specific recognition for certain carbohydrates. In order to construct an efficient specific recognition system, maltoside alkanethiol was adopted as an effective sensing modifier having a disaccharide group and a flexible long alkyl chain. The surface modification of gold nanoparticles with maltoside alkanethiol resulted in a shift and broadening (from 520 to 610 nm) of the absorption peak. Monodispersed maltoside-adsorbed gold nanoparticles aggregated with the specific lectin, concanavalin A (Con A). This phenomenon was used to detect the presence of Con A and to estimate concentrations of Con A in sample solutions. The precipitate of the maltoside–gold nanoparticle–Con A mixture was redispersed by addition of methyl α-D-mannopyranoside whose adsorption coefficient is larger than that of maltoside with Con A.     

Evaluation of concanavalin A-mannose interaction on the electrode covered with collagen film

An electrode covered with a lectin/collagen film was constructed to investigate whether the film was usable as a reaction field of binding between the lectin and sugar. The protein-sugar binding on cell surface plays an important role to various physiologic processes. The film is considered to be a cell surface, due to its biocompatibility. The immobilization of concanavalin A (Con A) which is one of proteins was attempted by an electrostatic interaction of the protonated functional groups of film to the negative charged Con A. The merit of this immobilization is that the interaction hardly causes any changes in the protein structure. Because Con A recognizes mannose moiety, the mannose was labeled with an electroactive compound. The binding was estimated from the changes of the electrode response based on the holding of electroactive moiety in the binding site of Con A to the mannose moiety. However, the electrode responses of glucose and galactose labeled with the same substance did not change. The result shows that Con A is immobilized on the film and combines with labeled mannose. Therefore, it is clear that the collagen film is suitable as the reaction field to evaluate the protein-sugar binding.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells.

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10(7) binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37 degrees C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Dynamic light scattering as an efficient tool to study glyconanoparticle-lectin interactions

Glyconanomaterials, an emerging class of bio-functional nanomaterials, have shown promise in detecting, imaging and targeting proteins, bacteria, and cells. In this article, we report that dynamic light scattering (DLS) can be used as an efficient tool to study glyconanoparticle (GNP)--lectin interactions. Silica and Au nanoparticles (NPs) conjugated with D-mannose (Man) and D-galactose (Gal) were treated with the lectins Concanavalin A (Con A) and Ricinus communis agglutinin (RCA(120)), and the hydrodynamic volumes of the resulting aggregates were measured by DLS. The results showed that the particle size grew with increasing lectin concentration. The limit of detection (LOD) was determined to be 2.9 nM for Con A with Man-conjugated and 6.6 nM for RCA(120) with Gal-conjugated silica NPs (35 nm), respectively. The binding affinity was also determined by DLS and the results showed 3-4 orders of magnitude higher affinity of GNPs than the free ligands with lectins. The assay sensitivity and affinity were particle size dependent and decreased with increasing particle diameter. Because the method relies on the particle size growth, it is therefore general and can be applied to nanomaterials of different compositions.     

A Bifunctional Spin Label for Ligand Recognition on Surfaces

In situ monitoring of biomolecular recognition, especially at surfaces, still presents a significant technical challenge. Electron paramagnetic resonance (EPR) of biomolecules spin‐labeled with nitroxides can offer uniquely sensitive and selective insights into these processes, but new spin‐labeling strategies are needed. The synthesis and study of a bromoacrylaldehyde spin label (BASL), which features two attachment points with orthogonal reactivity is reported. The first examples of mannose and biotin ligands coupled to aqueous carboxy‐functionalized gold nanoparticles through a spin label are presented. EPR spectra were obtained for the spin‐labeled ligands both free in solution and attached to nanoparticles. The labels were recognized by the mannose‐binding lectin, Con A, and the biotin‐binding protein avidin‐peroxidase. Binding gave quantifiable changes in the EPR spectra from which binding profiles could be obtained that reflect the strength of binding in each case.     

Bacterial detection using carbohydrate-functionalised CdS quantum dots: a model study exploiting E. coli recognition of mannosides

Mannose-coated CdS quantum dots (Man-QDs) were prepared in a facile aqueous, one-pot process that exploits the self-assembly of thiolated mannose in the presence of CdS under reducing conditions. The resulting ∼15 nm diameter nanoparticles produce an intense, broad luminescence emission centred at 550 nm. These Man-QDs induce luminescent aggregates of Escherichia coli which can be used to detect bacteria in cell suspensions containing as few as 10⁴E. coli per mL. The aggregation process is dependent on the E. coli cell surface FimH mannose-specific lectin. The recognition and subsequent detection of the E. coli using the Man-QD has been shown to be specific as aggregation does not occur either with an E. coli strain defective in the FimH lectin or with galactose-coated QDs.     

Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery

The development of targeted vehicles for systemic drug delivery relies on optimizing both the cell-targeting ligand and the physicochemical characteristics of the nanoparticle carrier. A versatile platform based on modification of gold nanoparticles with thiolated polymers is presented in which design parameters can be varied independently and systematically. Nanoparticle formulations of varying particle size, surface charge, surface hydrophilicity, and galactose ligand density were prepared by conjugation of PEG-thiol and galactose-PEG-thiol to gold colloids. This platform was applied to screen for nanoparticle formulations that demonstrate hepatocyte-targeted delivery in vivo. Nanoparticle size and the presence of galactose ligands were found to significantly impact the targeting efficiency. Thus, this platform can be readily applied to determine design parameters for targeted drug delivery systems.Modified gold nanoparticles are a suitable model for nanoparticle-based gene carriers.     

Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor.

A protocol based on saturation transfer difference (STD) NMR spectra was developed to characterize the binding interactions at an atom level, termed group epitope mapping (GEM). As an example we chose the well-studied system of galactose binding to the 120-kDa lectin Ricinus communis agglutinin I (RCA(120)). As ligands we used methyl beta-D-galactoside and a biantennary decasaccharide. Analysis of the saturation transfer effects of methyl beta-D-galactoside showed that the H2, H3, and H4 protons are saturated to the highest degree, giving evidence of their close proximity to protons of the RCA(120) lectin. The direct interaction of the lectin with this region of the galactose is in excellent agreement with results obtained from the analysis of the binding specificities of many chemically modified galactose derivatives (Bhattacharyya, L.; Brewer, C. F. Eur. J. Biochem. 1988, 176, 207-212). This new NMR technique can identify the binding epitope of even complex ligands very quickly, which is a great improvement over time-consuming chemical modifications. Efficient GEM benefits from a relatively high off rate of the ligand and a large excess of the ligand over the receptor. Even for a ligand like the biantennary decasaccharide with micromolar binding affinity, the binding epitopes could easily be mapped to the terminal beta-D-Gal-(1-4)-beta-D-GlcNAc (beta-D-GlcNAc = N-acetyl-D-glucosamine) residues located at the nonreducing end of the two carbohydrate chains. The binding contribution of the terminal galactose residue is stronger than those of the penultimate GlcNAc residues. We could show that the GlcNAc residues bind "edge-on" with the region from H2 to H4, making contact with the protein. Analysis of STD NMR experiments performed under competitive conditions proved that the two saccharides studied bind at the same receptor site, thereby ruling out unspecific binding.     

Highly Sensitive Determination of Concanavalin A Lectin Based on Silver-Enhanced Electrogenerated Chemiluminescence of Luminol

A highly sensitive bioassay based on silver-enhanced luminol electrogenerated chemiluminescence (ECL) is reported for the determination of concanavalin A lectin. A gold electrode modified with the mixed self-assembled monolayer of thiolated mannoside and mercaptohexanol was used to selectively capture a target lectin, concanavalin A, through the specific interaction between mannoside and concanavalin A. Mannoside-functionalized gold nanoparticles were further introduced to the opposite binding sites of the tetrameric concanavalin A to form a sandwich-type complex. Silver enhancement step was performed to coat the surface of mannose-stabilized gold nanoparticles with silver. The deposited silver was dissolved in an acidic solution and further neutralized. The resulting silver ions were finally detected with luminol electrogenerated chemiluminescence, in which the silver ions greatly enhanced the chemiluminescence intensity. The present electrogenerated chemiluminescence bioassay detected concanavalin A from 0.190 to 10.0?µg/mL (r²?=?0.999) with a detection limit of 0.146?µg/mL (signal to noise ratio?=?3), which is much lower compared to previously reported methods such as microgravimetry, surface plasmon resonance, and colorimetry. Furthermore, the present bioassay showed good selectivity over possible interfering lectin proteins.     

Synthesis and Characterization of Water‐Soluble Conjugated Glycopolymer for Fluorescent Sensing of Concanavalin A

A neutral polyfluorene derivative that contains 20 mol % 2,1,3‐benzothiadiazole (BT) is synthesized by Suzuki cross‐coupling polymerization. A cationic conjugated polymer A and an α‐mannose‐bearing polymer B are subsequently obtained through different post‐polymerization methods. As a result of the charged pendant groups or sugar‐bearing groups attached to the polymer side chains, both A and B show good water‐solubility. The titration of Concanavalin A (Con A) into polymer aqueous solution leads to different fluorescent responses for polymers A and B . Polymer A does not show any obvious fluorescence change upon interaction with Con A, whereas polymer B shows fluorescence increase in BT emission intensity when Con A is added. This is because of the specific interaction between α‐mannose and Con A, which induces polymer aggregation, and then facilitates energy transfer from the phenylene–fluorene segments to the BT units. A practical calibration curve ranging from 1 nM to 250 nM is obtained by correlating the changes in BT emission intensity with Con A concentration. The advantage of polymer B ‐based Con A macromolecular probe is that it shows signal increase upon Con A recognition, which is significantly different from other conjugated polymer‐based fluorescence quenching assays.     

One-step synthesis of amino-dextran-protected gold and silver nanoparticles and its application in biosensors

A sensitive method for the detection of the lectin protein concanavalin A (Con A) was developed using amino-dextran (AD)-protected gold (AD-Au) and silver nanoparticles (AD-Ag) as sensitive optical probes. The AD-Au and AD-Ag nanoparticles were synthesized by directly applying amino-dextran as a reductive and protective reagent. The size of the nanoparticles could be altered by changing the molar ratio of AD to the metal salt. The amino-dextran bound to Con A by forming a 4:1 Au-Con A complex at neutral pH, and the nanoparticles were induced to aggregate by Con A. The absorption intensity of the nanoparticles decreased linearly with as the Con A concentration was increased from 3.85×10^–8 to 6.15×10^–7 M. The Au-Con A complex was dissociated by the disaccharide isomaltose, which has a higher affinities for Con A than Au; this competitive strategy could also be used to detect similar types of saccharides.     

Dextran-coated CdSe quantum dots for the optical detection of monosaccharides by resonance light-scattering technique

A resonance light-scattering (RLS) detection method for saccharides was developed using dextran-coated CdSe quantum dots (dextran-CdSe-QDs) optical probes. The dextran-CdSe-QDs can be aggregated with concanavalin A (Con A), and the change in RLS intensity is used to monitor the extent of aggregation. The presence of glucose competitively binds with Con A, dissociating the Con A/dextran-CdSe-QDs complexes, affording the RLS intensity change and hence determining glucose concentrations in the range from a few to about 90 mM. Transmission electron microscopy was used to investigate the competitive interaction between glucose and dextran-CdSe-QDs with Con A. The competitive strategy could also be used to detect similar types of saccharides and the affinities of various monosaccharides for Con A increased in the order galactose?glucose < fructose < mannose. The proposed method was successfully applied to determine glucose in the human serum.

A resonance light-scattering (RLS) detection method for saccharides was developed using dextran-coated CdSe quantum dots (dextran-CdSe-QDs) optical probes. The dextran-CdSe-QDs were coupled to concanavalin A (Con A) to facilitate the aggregation of nanoparticles. The presence of glucose competitively binds with Con A, dissociating the Con A/dextran-CdSe-QDs complexes affording the RLS intensity change and hence determining glucose in the range from a few millimolar to about 90 mM. The proposed method was applied to the determination of glucose in human serum samples with satisfactory results.

     

Lectin-Triggered Aggregation of Glyco-Gold Nanoprobes for Activity-based Sensing of Hydrogen Peroxide by the Naked Eye

The purpose of this study was to develop a colorimetric assay for detecting hydrogen peroxide (H₂O₂) through a combination of using an aryl boronate (AB) derivative and gold nanoparticles (AuNPs). The unique optical property of AuNPs is applied to design a detection probe. The aggregation of AuNPs could be directly observed as a color change by the naked eye. A mannoside-boronate-sulfide ( MBS ) ligand was designed that contains an arylboronate (AB), a mannoside, and a thiol group. The thiol group bonds covalently with the surface of AuNPs to obtain MBS@AuNPs. The mannoside moiety recognizes concanavalin A (Con A), a lectin with four carbohydrate recognition sites that can specifically recognize the non-reducing end of an α-D-mannoside or α-D-glucoside structure. The AB structure on MBS first reacts with H₂O₂ and then inserts an oxygen atom in the B−H bond, which triggers intramolecular electron rearrangement to cleave the covalent bond, resulting in a MBSt mixture. The MBS or MBSt is then modified to citrate-coated AuNPs (c-AuNPs) to have MBS@AuNPs or MBSt@AuNPs. When the MBS@AuNPs are incubated with Con A, the Con A recognizes multiple mannosides on the surface of the MBS@AuNPs. Subsequently, the MBS@AuNPs aggregate and the solution's color changes from red to purple, but this color change does not occur in the case of MBSt@AuNPs. The phenomenon can be observed by the naked eye.     

Photoassisted Synthesis of Luminescent Mannose–Au Nanodots for the Detection of Thyroglobulin in Serum

We have employed mannose‐modified gold nanodots (Man–Au NDs) as a luminescence sensor for the detection of the thyroid‐cancer marker thyroglobulin (Tg) in homogeneous solutions. The luminescent Man–Au NDs are prepared through the reaction of 2.9 nm‐diameter gold nanoparticles (Au NPs) with 11‐mercapto‐3,6,9‐trioxaundecyl‐α‐D ‐mannopyranoside (Man‐RSH) under the irradiation of a light‐emitting diode (LED). We have found that the irradiation enhances the quantum yield (～11 %), alters the emission wavelength and lifetimes, and shortens the preparation time. A luminescence assay has been developed for Tg based on the competition between Tg and Man–Au NDs for the interaction with the concanavalin A (Con A). Because luminescence quenching of the Man–Au NDs by Con A is inhibited by Tg selectivity, we have obtained a highly sensitive and selective assay for Tg.     

Carbohydrate-protein interactions at interfaces: synthesis of thiolactosyl glycolipids and design of a working model for surface plasmon resonance

Thiolactosyl lipids designed for carbohydrate-protein binding studies have been synthesised. One representative was selected for binding studies with a plant lectin RCA120, the agglutinin from Ricinus communis. The interactions were measured quantitatively in real time using a BIAcore surface plasmon resonance instrument. Removal of much of the galactose from the thiolactosyl lipid in situ with beta-galactosidase showed that the lectin binding was highly specific. A dissociation constant KD = 8.77 x 10(-8) M was measured for 1-[2-[2-(2-[beta-D-galactopyranosyl-(1-->4)-1-thio-beta-D -glucopyranosyl]ethoxy)ethoxy]ethoxy]octadecane 30 which is four orders of magnitude greater than that determined for binding to lactose in solution. A concentration of lactose of > 80 mM was required to block the lectin binding to thiolactosyl lipid in a neomembrane.     

Self-organized glycoclusters along DNA: effect of the spatial arrangement of galactoside residues on cooperative lectin recognition

We describe herein the relationship between the spatial arrangement of self-organized galactose clusters and lectin recognition. beta-Galactose-modified deoxyuridine phosphoramidite was synthesized and applied to solid-phase synthesis to provide 18-, 20-, and 22-mers of site-specifically galactosylated oligodeoxynucleotides (Gal-ODNs). These Gal-ODNs were self-organized through hybridization with the corresponding 18-, 20-, and 22-mers of half-sliding complementary ODNs (hsc-ODNs) to give periodic galactoside clusters. The self-organization of ODNs was confirmed by size exclusion chromatography and gel electrophoresis. The binding of the Gal-clusters to the FITC-labeled RCA(120) lectin was analyzed by monitoring the change in fluorescence intensity. The assembly of 20-mer Gal-ODN with the 20-mer hsc-ODN was strongly and cooperatively recognized by the lectin. The 18-mer assembly was bound more weakly and less cooperatively, and the 22-mer assembly was minimally bound to the lectin. RCA(120) lectin recognized not only the density of galactoside residues, but also the spatial arrangement. The size of the Gal cluster was estimated from the association constant of Gal-ODN with hsc-ODN. The relationship between lectin-recognition and Gal-cluster size is also discussed.