Immunofunctionalisation of gold transducers for bacterial detection by physisorption

Functionalisation of the sensing surface is a key factor in immunosensor fabrication as it allows target-selective capture and prevents nonspecific adsorption of undesired components. Gold immunofunctionalisation using self-assembled monolayers (SAM) has been widely exploited to this end for the detection of small targets. However, we recently demonstrated that this strategy fails when detecting whole bacteria cells (Baldrich et al., Anal Bioanal Chem 390:1557–1562, 2008). We now investigate different physisorption-based alternatives using E. coli as the target organism. Our results demonstrate that physisorption generates the appropriate substrate for the specific detection of bacteria on gold surfaces, providing detection limits down to 10⁵ cells mL⁻¹ in an ELISA-type colorimetric assay. Additionally, surface coverage is highly reproducible when assayed by impedance spectroscopy and the inter- and intra-assay coefficients of variation are below 10–15% in all cases. These surfaces were stable, retained functionality and did not suffer from significant biomolecule desorption after 10 days storage in PBS at 37 °C, hence confirming physisorption as a cheap, simple and efficient strategy for the detection of bacteria.     

Quantitative imaging of protein adsorption on patterned organic thin-film arrays using secondary electron emission

Secondary electron emission is developed as a means to quantify and image protein binding to Au surfaces modified with patterned organic thin-film arrays. Alkane thiols were patterned via microcontact printing on gold, and their effects on the secondary electron (SE) yield of the surface, systematically quantified. We show that a self-assembled monolayer (SAM) of hexadecane thiol significantly increases the SE yield over the native gold surface, a yield that increases as a function of alkane chain length (C8-C16). This effect is linearly correlated with the surface potentials and wetting properties of these SAMs. Surface layers comprised of poly(ethylene glycol) (PEG) grafted polyacrylamide polymers behave differently, affecting the SE yield by attenuation according to the polymer thickness. These results demonstrate the relative contributions of factors related to the adsorbate molecular structures that serve to strongly mediate the SE yield, providing a foundation for exploiting them as a quantitative electron imaging probe. The latter capability is demonstrated using a model microfluidic assay in which a series of proteins was spatially addressed to a SAM-based pixel array. The gray scale contrasts seen with protein adsorption are directly correlated with both protein molecular weight and mass coverage. These methods are used in two model protein assay experiments: (1) the measurement of the concentration dependent adsorption isotherm for a model protein (fibrinogen); and (2) the selective recognition of a biotinylated protein layer by avidin. These results demonstrate a unique approach to imaging protein binding processes on surfaces with both high analytical and spatial sensitivity.     

Fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate: a reagent for formation of interchain carboxylic anhydrides on self-assembled monolayers

In this paper, we report the reactivity of fluoro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TFFH), a reagent for transformation of carboxylic acids into acid fluorides in solution, toward self-assembled monolayers (SAMs) of 16-mercaptohexadecanoic acid on gold. Contrary to the solution-based reactions, we found that only interchain carboxylic anhydrides (ICAs), not acid fluorides (AFs), were obtained at surfaces by the facile interchain reaction under most reaction conditions studied. AFs were found to be formed only when tetrabutylammonium fluoride, a reagent inducing fast decomposition of ICAs, was added to the reaction mixture. The reactivity of TFFH toward carboxylic acid-terminated SAMs was different from that of cyanuric fluoride, which has been reported previously (Langmuir 2005, 21, 11765-11772). This study provides more insight into the role of the proximity effect in SAM-based reactions as well as another approach to the formation of ICAs from carboxylic acid-terminated SAMs.     

Steric crowding effects on target detection in an affinity biosensor

This work quantifies the impact of steric crowding on whole antibody (Ab) receptor immobilization and target Ab detection and also demonstrates how the versatile biotin/streptavidin receptor immobilization system must be tuned to optimize target detection in designing biosensors. Results are demonstrated on a label-free optical biosensor fabricated from n-type macroporous porous silicon (PSi) with approximately 88-107 nm diameter pores. We employ a sandwich assay scheme comprising a linking chemistry (biotin/streptavidin) to attach biotinylated anti-rabbit IgG (receptor) to detect rabbit IgG (target). A "bottom-up" approach was taken to investigate each layer of the sandwich assay to optimize target binding. Steric crowding was observed to hinder subsequent layer binding for each layer in the sandwich (biotin, streptavidin, and receptor). Our results give definitive evidence that the onset of steric crowding within the biotin layer occurs at a surface coverage of 57%, which is much higher compared to that from published work on well-ordered self-assembled biotin monolayers on planar gold surfaces. This difference is attributed to the topographical heterogeneity of the PSi substrate. Streptavidin (SA) binding to surface-linked biotin was altered by preblocking the streptavidin binding sites with biotin. Through consistent trends in data, preblocking SA was shown to reduce steric crowding within the SA layer, which translated into increased receptor immobilization. The final detection range of rabbit IgG was 0.07-3 mg mL(-1) (0.4-17 ng mm(-2)), and binding specificity was demonstrated by employing an anti-chicken IgG control receptor. This study underlines the importance of considering binding avidity and surface topography in optimizing chip-based biosensors.     

Restricted surface mobility of thiolate-covered metal surfaces: a simple strategy to produce high-area functionalized surfaces

We have studied the self-assembly of thiol monolayers on high-area nanostructured gold surfaces. These surfaces are highly irregular with a fractal dimension close to 2.5. Auger electron spectroscopy and voltammetric data indicate that thiol self-assembly with a maximum surface coverage approximately 1/3 takes place, the same result as that found for smooth gold surfaces. Therefore, neither curvature effects, which would promote higher coverage, nor excluded volume effects, which would result in lower coverage, are present in these irregular surfaces. The high surface area of the bare electrodes exhibits a rapid surface decay in different liquid media that is hindered by alkanethiolate chemisorption. The presence of thiolate SAMs reduces markedly the mass transport surface diffusion of gold adatoms, hindering surface area decay and freezing the system in a metastable state for days. This effect cannot be explained by considering only hydrocarbon-hydrocarbon chain interactions, because it is also observed for ordered arrays of adsorbed S atoms. Therefore, interactions between ordered chemisorbed species at high coverage seem to be responsible for the observed behavior. The thiol-covered high-area metallic substrates can be used to efficiently anchor a large number of molecules, biomolecules, or nanostructures, improving the performance of SAM-based optical and electrochemical devices.     

Arrays for the combinatorial exploration of cell adhesion

A new method for the fabrication of arrays of self-assembled monolayers (SAMs) of alkane thiols (ATs) on gold to combinatorially assay surfaces for cell adhesion is reported. A fluorous SAM, which is both cytophobic and solvophobic, was used as the background between the array features. The resulting solvophobic background permits the application of an assembly after conjugation strategy for fabrication. SAMs containing mixtures of ATs and peptide-terminated ATs were generated. Multiple cell types demonstrated differential and specific binding to these surfaces. Additionally, pluripotent human embryonic stem cells proliferated on surfaces generated by this method.     

Staphylococcus aureus adhesion to self-assembled monolayers: effect of surface chemistry and fibrinogen presence

Staphylococcus aureus adhesion on self-assembled monolayers (SAMs) formed by the adsorption of alkanethiols on transparent gold films has been studied in real time under well-defined flow conditions using a radial flow chamber and an automated videomicroscopy system. SAMs terminated with methyl, hydroxyl, carboxylic acid and tri(ethylene oxide) groups were investigated. SAMs were characterized using contact angle measurements, ellipsometry and X-ray photoelectron spectroscopy. Adhesion experiments using the Newman strain of S. aureus were performed on bare monolayers and monolayers pre-incubated with fibrinogen. Adhesion was found to be lowest on the ethylene oxide-bearing surfaces, followed by the hydroxyl surfaces. Adhesion on the carboxylic- and methyl-terminated SAMs was much higher. Bacterial adhesion was higher on the hydrophobic surfaces. Pre-incubation of surfaces with fibrinogen minimized the effect of the surface properties of the substrate. Adhesion was increased on all surfaces when fibrinogen was present and no significant differences were observed between adhesion to the different SAMs. This study showed that surfaces rich in ethylene oxide groups can be effectively used to prevent bacterial adhesion. However, under physiological conditions, most of the substrate properties are masked by the presence of the adsorbed protein layer and the effect of substrate properties on bacteria adhesion under flow is minimal.     

A simple method of surface functionalisation for immuno-specific immobilisation of proteins

We present a new and advanced methodology, developed for surface functionalisation of gold and to study immobilisation of an immuno-specific system of proteins. A combination of electrochemical quartz crystal microbalance and Raman spectroscopy techniques allowed a complete understanding of the system starting from surface functionalisation and progressing to the functional structure analysis of immobilised proteins. A simple electrochemical procedure was formulated to prepare sulphonyl chloride terminated gold surfaces that form a strong sulphonamide bond with the receptor protein staphylococcal protein A (SpA). On the SpA grafted surfaces, the immobilisation of a human IgG and consecutive binding of an immuno-specific anti-human IgG was observed. The surface functional groups form a strong interaction with SpA without disturbing its functional properties. The native functional structure of SpA and also the IgGs was found to be retained in their immobilised state.     

Development and validation of a sensitive and fast chemiluminescent enzyme immunoassay for the detection of genetically modified maize

Proteins from the Cry 1 family, in particular Cry 1Ab, are commonly expressed in genetically modified Bt maize in order to control chewing insect pests. A sensitive chemiluminescent sandwich enzyme immunoassay for the detection of Cry1Ab protein from genetically modified Bt maize has been developed and validated. A Cry1Ab protein-specific antibody was immobilized on 96- or 384-well microtiter plates in order to capture the Cry1Ab toxin in the sample; the bound toxin was then detected by employing a second anti-Cry1Ab antibody and a horseradish peroxidase-labeled anti-antibody, followed by measurement of the enzyme activity with an enhanced chemiluminescent system. The chemiluminescent assay fulfilled all the requirements of accuracy and precision and exhibited limits of detection of a few pg mL⁻¹ Cry1Ab (3 or 5 pg mL⁻¹, depending on the assay format), which are significantly lower than that achievable using conventional colorimetric detection of peroxidase activity and also represent an improvement compared to previously developed Cry1Ab immunoassays. High-throughput analysis can be performed using the 384-well microtiter plate format immunoassay, which also allows one to reduce the consumption of samples and reagents. Validation of the assay, performed by analyzing certified reference materials, proved that the immunoassay is able to detect the presence of the Cry1Ab protein in certified reference samples containing as low as 0.1% of MON 810 genetically modified Bt maize. This value is below the threshold requiring mandatory labeling of foods containing genetically modified material according to the actual EU regulation.     

Electrochemically deposited palladium as a substrate for self-assembled monolayers

The vast majority of reports of self-assembled monolayers (SAMs) on metals focus on the use of gold. However, other metals, such as palladium, platinum, and silver offer advantages over gold as a substrate. In this work, palladium is electrochemically deposited from PdCl2 solutions on glassy carbon electrodes to form a substrate for alkanethiol SAMs. The conditions for deposition are optimized with respect to the electrolyte, pH, and electrochemical parameters. The palladium surfaces have been characterized by scanning electron microscopy (SEM) and the surface roughness has been estimated by chronocoulometry. SAMs of alkane thiols have been formed on the palladium surfaces, and their ability to suppress a Faradaic process is used as an indication for palladium coverage on the glassy carbon. The morphology of the Pd deposit as characterized by SEM and the blocking behavior of the SAM formed on deposited Pd delivers a consistent picture of the Pd surface. It has been clearly demonstrated that, via selection of experimental conditions for the electrochemical deposition, the morphology of the palladium surface and its ability to support SAMs can be controlled. The work will be applied to create a mixed monolayer of metals, which can subsequently be used to create a mixed SAM of a biocomponent and an alkanethiol for biosensing applications.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.     

Highly sensitive detection of protein and small molecules based on aptamer-modified electrochemiluminescence nanoprobe

Amplified optical detection of biomolecules using nanoparticle as the carrier has attracted considerable interest in the scientific community. In this study, a promising aptasensor was developed for highly sensitive detection of protein and small molecules based on the construction of aptamer-modified electrochemiluminescence (ECL) nanoprobe. Specifically, thrombin and ATP serve as the examples for detection. By taking advantage of sandwich binding of two affinity aptamers for high specificity, tris-(2,2'-bipyridyl)ruthenium (TBR)-cysteamine loaded in gold nanoparticle (GNP) as barcodes for signal amplification, and micromagnetic particles (MMPs) based ECL technology for rapid detection, a novel assay for biomolecules quantification was developed. The sandwich complex containing targets could be selectively captured by MMPs and then quantified by ECL intensity. We have demonstrated that the detection limits of human thrombin and ATP are 1 pM and 10 pM, respectively, with high specificity. The proposed technology is expected to become a powerful tool for biomolecule analysis.     

Single particle technique for one-step homogeneous detection of cancer marker using gold nanoparticle probes

In this paper, we reported a single particle technique for the one-step homogeneous immunoassay of a cancer marker by resonance light scattering correlation spectroscopy (RLSCS). The setup of RLSCS was similar to fluorescence correlation spectroscopy (FCS), and its principle was based on measuring the resonance light scattering fluctuations in a small volumes (less than 1 fL) due to Brownian motion of single particles. In homogeneous immunoassay, we used a sandwich strategy and conjugated two different antibodies (Ab) with gold nanoparticles (GNPs) respectively. When two different GNPs labeled with antibodies are mixed in a sample containing antigen (Ag) targets, the binding of targets will cause GNPs to form dimers (or oligomers), which leads to the significant increase in the characteristic diffusion time of GNPs in the detection volume. The RLSCS method can sensitively detect the change in the characteristic diffusion time of GNPs before and after immune reactions. We used this technology in homogeneous immunoassays for the liver cancer biomarker alpha-fetoprotein (AFP). The conditions of the immune reaction were investigated systematically. In the optimal conditions, the linear range of this assay is from 1 pM to 1 nM and the detection limit is 1 pM for AFP. This new method was successfully applied for the direct determination of AFP levels in sera from healthy subjects and cancer patients. Our results were in good agreement with ELISA assays.     

Development of dipsticks for simultaneous detection of vip3A and cry1Ab/cry1Ac transgenic proteins

The number of genetically modified (GM) crops being cultivated and its produce reaching market is increasing every year. The transgenes (vip3A, cry1Ab, and cry1Ac) from Bacillus thuringiensis are being used by plant breeders, apart from other transgenes for developing insect pest-resistant GM crops. It is therefore necessary to develop an easy, rapid, and reliable detection assay to discriminate GM crops and non-GM crops. Dipstick strips using colloidal gold-labeled polyclonal antibodies were developed for simultaneous detection of Vip3A and Cry1Ab/CrylAc proteins. The assay was essentially based on the sandwich format of immunoassay, which was completed within 10 min, and the results were evaluated visually. The detection limits were 50 ng/mL (50 ppb) for both CrylAc and CrylAb proteins, and 100 ng/mL (100 ppb) for Vip3A protein. The developed dipsticks are suitable for on-site simultaneous screening of GM crops bearing two proteins, which, in turn, reduce cost and time of the assay.     

Oriented protein adsorption to gold nanoparticles through a genetically encodable binding motif

Simple, stable, and specific methods for immobilizing proteins on gold surfaces are needed for the development of applications that rely on the oriented attachment of proteins to gold surfaces. We report a direct, stable, genetically encodable method for the oriented chemisorption of proteins to gold nanoparticles (Au NPs) through the tetracysteine motif (C-C-P-G-C-C) while simultaneously suppressing protein physisorption. Mutants of ubiquitin (Ub) and enhanced green fluorescent protein (eGFP) containing the tetracysteine motif were produced and displayed stronger adsorption to the NPs than did native proteins. An eGFP mutant with a dicysteine motif (G-C-C) did not show a significant improvement in binding to Au NPs compared to that of the wild-type protein. The binding of the proteins to Au NPs of various sizes (14, 18, 28, and 39 nm) was explored. The small Ub tetracysteine mutant stabilized several sizes of Au NPs, and the eGFP tetracysteine mutant clearly had the strongest chemisorption to the 18 nm NPs. The control of binding orientation for proteins bearing a tetracysteine motif was demonstrated through the enhanced specific binding of protein-NP conjugates to immobilized targets.     

Stopped-flow microarray immunoassay for detection of viable <Emphasis Type="Italic">E. coli</Emphasis> by use of chemiluminescence flow-through microarrays

Rapid multiplexed analysis of microorganisms is important in water analysis to control bacterial contamination for health and safety reasons. Direct quantification of bacteria by means of flow-through microarray immunoassays requires new analysis strategies for optimising sensitivity and the analysis time. For bacteria and for particles, hydrodynamic forces and sedimentation are the dominating effects for binding on surfaces in a flow-through system, whereas diffusion is insignificant. Therefore, we have implemented a stop and flow technique for quantification of viable E. coli cells. The method, with alternation of resting volume elements and pumping the elements forward, was more effective than continuous-flow approaches for analysis of bacteria. For quantification of viable E. coli cells, a chemiluminescence sandwich immunoassay test format was performed by means of antibody microarrays and flow-injection-based microarray analysis. Antibodies, which served as selective capture molecules, were immobilised on polymer-modified glass surfaces serving as microarray substrate. For the bacteria recognition step, a second detection antibody was used, forming a sandwich immunoassay at each spot of the microarray. Detection was carried out with a horseradish peroxidase catalysed chemiluminescence reaction. All assay steps were conducted with an automated flow-through chemiluminescence microarray readout system. Living E. coli cells could be detected in 67 min with a detection limit of 4 × 10⁵ cells mL⁻¹. By introduction of the stopped-flow technique and optimisation of interaction time and interaction steps the achieved detection of E. coli was faster and two orders of magnitude more sensitive than with a conventional ELISA technique in microplates.     

Preparation of high quality electrical insulator self-assembled monolayers on gold. Experimental investigation of the conduction mechanism through organic thin films

Self-assembled monolayers (SAMs) form highly ordered, stable dielectrics on conductive surfaces. Being able to attach larger-area contacts in a MIM (metal-insulator-metal) diode, their electrical properties can be determined. In this paper, the electrical conduction through thiolate SAMs of different alkyl chain lengths formed on gold surfaces were studied and discussed. The influence of the headgroup with respect to the surface quality and prevention of short circuits is investigated. Phenoxy terminated alkanethiols were found to form high quality SAMs with perfect insulating properties. Synthesis of the required terminally substituted long chain thiols have been developed. The I(V) characteristics of MIM structures formed with these SAMs are measured and simulated according to theoretical tunneling models for electrical conductivity through thin organic layers. SAM based electronic devices will become especially important for future nanoscale applications, where they can serve as insulators, gate dielectric of FETs, resistors, and capacitor structures.     

Characterization of troponin T binding aptamers for an innovative enzyme-linked oligonucleotide assay (ELONA)

Early diagnosis of acute myocardial infarction (AMI) is of outmost importance to reduce the mortality rate, and cardiac troponins are considered the gold standard biomarkers of myocardial necrosis. In this scenario, the characterization of two troponin T (TnT)-binding aptamers as viable alternative to antibodies employed on clinical immunoassays is here reported for the first time. Their recognition ability was first investigated through surface plasmon resonance (SPR). Subsequently, an enzyme-linked oligonucleotide assay (ELONA) was developed on common 96-well polystyrene plates, both by direct and sandwich detection strategies for comparison. In both cases, the assay exhibits a detection ability of TnT in the range of low nanomolar but a great advantage on serum interference was obtained by using both aptamers in a sandwich format, with excellent reproducibility and recovery values. Despite the sensitivity needing to be enhanced to the low picomolar range, these results are encouraging for the development of new, low-cost, and rapid antibody-free colorimetric assays for AMI studies based on aptamer–Troponin T recognition.

     

Self-assembly of alkanols on Au(111) surfaces

Self-assembled monolayers (SAMs) of alkanols (1-C(N)H(2N+1)OH) with varying carbon-chain lengths (N = 10-30) have been systematically studied by means of scanning tunneling microscopy (STM) at the interfaces between alkanol solutions (or liquids) and Au(111) surfaces. The carbon skeletons were found to lie flat on the surfaces. This orientation is consistent with SAMs of alkanols on highly oriented pyrolytic graphite (HOPG) and MoS2 surfaces, and also with alkanes on reconstructed Au(111) surfaces. This result differs from a prior report, which claimed that 1-decanol molecules (N = 10) stood on their ends with the OH polar groups facing the gold substrate. Compared to alkanes, the replacement of one terminal CH3 group with an OH group introduces new bonding features for alkanols owing to the feasibility of forming hydrogen bonds. While SAMs of long-chain alkanols (N > 18) resemble those of alkanes, in which the aliphatic chains make a greater contribution, hydrogen bonding plays a more important role in the formation of SAMs of short-chain alkanols. Thus, in addition to the titled lamellar structure, a herringbone-like structure, seldom seen in SAMs of alkanes, is dominant in alkanol SAMs for values of N < 18. The odd-even effect present in alkane SAMs is also present in alkanol SAMs. Thus, the odd N alkanols (alkanols with an odd number of carbon atoms) adopt perpendicular lamellar structures owing to the favorable interactions of the CH3 terminal groups, similar to the result observed for odd alkanes. In contrast to alkanes on Au(111) surfaces, for which no SAMs on an unreconstructed gold substrate were observed, alkanols are capable of forming SAMs on either the reconstructed or the unreconstructed gold surfaces. Structural models for the packing of alkanol molecules on Au(111) surfaces have been proposed, which successfully explain these experimental observations.     

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.