A comparative study of capacitive immunosensors based on self-assembled monolayers formed from thiourea, thioctic acid, and 3-mercaptopropionic acid

A procedure was developed for the covalent coupling of anti-alpha-fetoprotein antibody (anti-AFP) to a gold surface modified with a self-assembled monolayer (SAM) of thiourea (TU). The performance of the SAM-antibody layer was compared to those of similar layers based on thioctic acid (TA) and 3-mercaptopropionic acid (MPA) by using flow injection capacitive immunosensor system. Covalent coupling of anti-AFP on self-assembled thiourea monolayer (SATUM) modified gold electrode can be used to detect alpha-fetoprotein with high efficiency, similar sensitivity, the same linear range (0.01-10 microgl(-1)) and detection limit (10 ngl(-1)) as those obtained from sensors based on self-assembled thioctic acid monolayer (SATAM) and self-assembled 3-mercaptopropionic acid monolayer (SAMPAM). The system is specific for alpha-fetoprotein and can be regenerated and reused up to 48 times. Therefore, self-assembled monolayer using thiourea which is cheaper than thioctic acid and 3-mercaptopropionic acid is a good alternative for biosensor applications when SAMs are used.     

Highly sensitive electrochemical detection of immunospecies based on combination of Fc label and PPD film/gold nanoparticle amplification

A highly sensitive electrochemical immunoassay strategy based on the combination of ferrocene (Fc) label and poly(o-phenylenediamine) (PPD) film/gold nanoparticle (GNP) amplification for the detection of immunospecies is proposed using human IgG as the model analyte. A gold electrode is firstly modified with an electropolymerized film of poly(o-phenylenediamine), which provides a stable matrix with abundant amino-groups for the fabrication of sensing interface. Using glutaraldehyde as a cross-linker, cystamine is coupled onto the modified electrode. Subsequently, gold nanoparticle monolayer is assembled onto the resulting surface. Making use of the unique properties of gold nanoparticles, antibodies can be self-assembled onto the surface-confined gold nanoparticles via amine-Au affinity with a high loading amount and reserve high immunological activity. After the introduction of model analyte, the ferrocene (Fc)-labeled antibody is immobilized on the sensing interface by antibody-antigen specific reaction, resulting in a redox current signal. The peak current is proportional to the amount of the analyte. Under the optimized experimental conditions, the proposed sensing strategy provides a wide linear dynamic range from 25 to 1000pg/mL with a low detection limit of 10pg/mL. In addition, good reproducibility, high selectivity and stability are achieved. In particular, the extremely high stability of both poly(o-phenylenediamine) and gold nanoparticle monolayer allows the designed biosensing interface to withstand harsh regeneration treatment, making it reusable.     

Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer

We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level.     

Binding of vancomycin group antibiotics to D-alanine and D-lactate presenting self-assembled monolayers

Peptides terminating in -Lys-D-Ala-D-Ala, -Lys-D-Ala-L-Ala and -Lys-D-Ala-D-Lactate were covalently coupled via an N-terminal aminohexanoic acid linker to a self-assembled monolayer of HS(CH2)15CO2H on a thin gold film. Binding of the glycopeptide antibiotics vancomycin and chloroeremomycin to these surfaces was then measured using a surface plasmon resonance biosensor. Both antibiotics bound with micromolar affinity to the D-Ala-terminating surface and with millimolar affinity to the D-Lactate-terminating surface. Increasing density of these covalently attached peptides on the surface had no effect on the resultant affinities of either antibiotic for the surface. In contrast, when the lipid-anchored peptide N-alpha-docosanoyl-epsilon-acetyl-Lys-D-Ala-D-Ala was inserted into a supported lipid monolayer, the affinity of the strongly dimerizing antibiotic chloroeremomycin for the surface showed a dependence on ligand density. This was not the case with the weakly dimerizing antibiotic vancomycin. The lipid monolayer surface, which is a more realistic model of the surface of a bacterium, was thus better suited for the study of the cooperative binding interactions that occur between dimeric glycopeptide antibiotics and surface-bound ligands.     

Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.     

A high sensitivity amperometric biosensor using a monomolecular layer of laccase as biorecognition element

Laccases from various sources were tested, and laccase from Rigidoporus lignosus was found to be the most active towards syringaldazine and ABTS, which are typical substrates of this class of enzymes, and towards the phenols found in olive oil mill wastewaters. This laccase was covalently immobilised by carbodiimide chemistry, on a self-assembled monolayer of 3-mercaptopropionic acid deposited on a gold surface. A flow biosensor, using the monolayer of laccase as bioelement and a glassy carbon electrode as amperometric transduction system, was developed. Although the amount of the immobilised enzyme (about 140 ng/cm2 effective surface area) was tiny, the biosensor showed a sensitivity of 3 nA/microM when 1,4-hydroquinone was used as substrate, and a half-life of 35 days. The proposed device permits detection of phenols in aqueous solutions at concentrations in the low micromolar range, i.e. below European Community limits. The biosensor was successfully used to detect phenols in wastewaters from an olive oil mill after minimal sample preparation (incubation of the aqueous sample with sodium borohydride for a few minutes) to suppress the current due to oxidised compounds present in the wastewaters.     

Anti-rabbit immunoglobulin G detection in complex medium by PM-RAIRS and QCM Influence of the antibody immobilisation method

Two antibody immobilisation procedures were compared to set up an immunosensor for goat anti-rabbit immunoglobulin (anti-rIgG), i.e. rIgG covalently bound or immobilised via affinity to protein A (PrA). In both cases, the first layer of protein was covalently bound to a mixed self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) and mercaptohexanol (C6OH) on a gold surface. The elaboration of the sensitive surfaces, as well as their selectivity and sensitivity were studied step by step by polarization modulation-reflection absorption infra-red spectroscopy (PM-RAIRS) and quartz crystal microbalance (QCM) with impedance measurement. QCM measurements showed that the viscoelastic properties of the antibody layer were markedly modified during the antigen recognition when the antibody was bound by affinity to PrA. The specific detection of antigen within a complex medium was assessed by PM-RAIRS thanks to the grafting of cobalt-carbonyl probes. Affinity constants between the immobilised rIgG and the anti-rIgG were determined from PM-RAIRS analysis.     

Single-step perfusion chromatography with a throughput potential for enhanced peptide detection by matrix-assisted laser desorption/ ionization-mass spectrometry

Mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis can be routinely performed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) which has become a standard tool. Since MALDI-MS detection relies heavily on the quality of the MALDI target, development of an efficient sample preparation technique for removal of sample contaminants is necessary. To date, among the several sample preparation techniques for MALDI targets available, multistep perfusion chromatography (MSPC) using Poros R2 and Oligo R3 has been most commonly used. However, MSPC requires at least four working steps and is not efficient for high-throughput analysis and recovery of low abundance proteins. During the course of proteomic analysis of a large set of rat liver tissues and the immortalized human sebaceous gland cells (SZ95 cells), we were interested in developing an alternative to MSPC. Here, we describe a single-step perfusion chromatography (SSPC) method for MALDI target preparation, which uses a tiny column packed with a mixture of Poros R2 and Oligo R3 resins. The SSPC method significantly improves not only detection of peptides but also efficiency of sample handling, thus enabling high-throughput sample preparation for analyzing large set of samples with high resolution and reproducibility.     

Detection of estradiol at an electrochemical immunosensor with a Cu UPD|DTBP-Protein G scaffold

A copper monolayer was formed on a gold electrode surface via underpotential deposition (UPD) method to construct a Cu UPD|DTBP-Protein G immunosensor for the sensitive detection of 17β-estradiol. Copper UPD monolayer can minimize the non-specific adsorption of biological molecules on the immunosensor surface and enhance the binding efficiency between immunosensor surface and thiolated Protein G. The crosslinker DTBP (Dimethyl 3,3'-dithiobispropionimidate · 2HCl) has strong ability to immobilize Protein G molecules on the electrode surface and the immobilized Protein G provides an orientation-controlled binding of antibodies. A monolayer of propanethiol was firstly self-assembled on the gold electrode surface, and a copper monolayer was deposited via UPD on the propanethiol modified electrode. Propanethiol monolayer helps to stabilize the copper monolayer by pushing the formation and stripping potentials of the copper UPD monolayer outside the potential range in which copper monolayer can be damaged easily by oxygen in air. A droplet DTBP-Protein G was then applied on the modified electrode surface followed by the immobilization of estradiol antibody. Finally, a competitive immunoassay was conducted between estradiol-BSA (bovine serum albumin) conjugate and free estradiol for the limited binding sites of estradiol antibody. Square wave voltammetry (SWV) was employed to monitor the electrochemical reduction current of ferrocenemethanol and the SWV current decreased with the increase of estradiol-BSA conjugate concentration at the immunosensor surface. Calibration of immunosensors in waste water samples spiked with 17β-estradiol yielded a linear response up to ≈ 2200 pg mL(-1), a sensitivity of 3.20 μA/pg mL(-1) and a detection limit of 12 pg mL(-1). The favorable characteristics of the immunosensors such as high selectivity, sensitivity and low detection limit can be attributed to the Cu UPD|DTBP-Protein G scaffold.     

Analysis of type I signal peptidase affinity and specificity for preprotein substrates

Type I signal peptidases (SPases) are membrane-bound endopeptidases responsible for the catalytic cleavage of signal peptides from secretory proteins. Here, we analysed the interaction between a bacterial type I SPase and preprotein substrates using surface plasmon resonance. The use of a home-made biosensor surface based on a mixed self-assembled monolayer of thiols on gold allowed qualitative and kinetic analysis. In vitro binding of purified preproteins to a covalently immobilised bacterial SPase was found to be rather efficient (apparent K(D)=10(-7)-10(-8)M). The signal peptide was shown to be a prerequisite for SPase binding and the nature of the mature part of the preprotein significantly affected SPase binding affinity. The developed biosensor containing immobilised SPase is of great importance for analysis of specificity at substrate binding level and for drug screening. In fact, this is the first report of a membrane protein that was covalently attached to a biosensor surface and that retained binding capacity.     

Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous sample collection and sample handling protocols. The sensitivity of the MS analysis relies on the quality of the sample; consequently, the blood sample preparation step is crucial to obtain pure and concentrated samples and enrichment of the proteins and peptides of interest. This review focuses on the serum sample preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE sample preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting.     

On‐target and nanoparticle‐facilitated selective enrichment of peptides and proteins for analysis by MALDI‐MS

Over the course of the last decade, a number of investigators have come to appreciate that the surface of a MALDI target, after suitable modification, can be used for selective enrichment of peptides and proteins. More recently, surface‐modified nanoparticles (NPs) that readily co‐crystallize in MALDI matrix, are not ionized by laser desorption/ionization, and do not interfere with MS have attracted interest as alternatives to surface‐modified targets for selective enrichment of peptides and proteins. Surface‐modified targets and NPs facilitate parallel processing of samples, and when used in conjunction with MALDI mass spectrometers with kHz lasers enable development of high‐throughput proteomics platforms. Targets and NPs for reversed phase and ion exchange retention, selective enrichment of glycopeptides, selective enrichment of phosphopeptides, and immunoaffinity MS are described in conjunction with details regarding their preparation and utility. Commercial availability of the reagents and substrates required to prepare surface‐modified targets and NPs is also discussed.     

Simple and robust two-layer matrix/sample preparation method for MALDI MS/MS analysis of peptides

Recent advances in MALDI MS/MS instrumentation allow a high degree of automation in the efficient detection of peptide fragment ions that can be used for protein identification. However, the performance of the technique is dependent on the MALDI sample preparation. We present a simple and robust two-layer sample preparation method tailored for sensitive and reproducible generation of MALDI MS/MS data. This method produces a strong and uniform crystal layer which allows acquisition of high quality MS/MS spectra over the entire sample surface area. Furthermore, due to its crystal strength, the matrix/sample layer can be washed extensively on target, enabling direct analysis of samples containing impurities, such as salts and surfactants. This method is demonstrated to be very useful in routine analysis of in-gel tryptic digests of silver-stained protein gel spots, without the need of desalting steps or hunting for "hot" spots. As an example, seven threonine-phosphorylated proteins involved in signal transduction in response to growth factor stimulation within the lipid raft fractions of the IMR5 neuroblastoma cells have been identified using differential gel display, in-gel digestion and MALDI MS/MS with the new two-layer sample preparation method. Some of these proteins have the functions of maintaining raft structure or cell signaling.     

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: sample preparation, data analysis, and application to the analysis of complex peptide mixtures

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.     

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.     

Ultra trace analysis of small molecule by label-free impedimetric immunosensor using multilayer modified electrode

A multilayer electrode modified with a self-assembled thiourea monolayer (SATUM) followed by gold nanoparticles (AuNPs), mercaptosuccinic acid (MSA) and antibody was investigated for the detection of ultra trace amount of a small molecule (chloramphenicol) in an impedimetric system. The formation of the antibody-antigen complex at the electrode surface caused the impedance to increase. Under optimum conditions three modified electrodes were compared the SATUM/AuNPs/MSA electrode provided a wide linear range (0.50-10) × 10?1? M, and a very low determination limit of 1.0 × 10?1? M. This determination limit was much lower than the SATUM/AuNPs electrode, 1.0 × 10?1? M, and SATUM electrode, 4.7 × 10?1? M. The modified electrode provided good selectivity for chloramphenicol detection and can be reused up to 45 times with a relative standard deviation of lower than 4%. When applied to determine chloramphenicol in shrimp samples, the results agreed well with those obtained by the high-performance liquid chromatography coupled with a photo diode array detector (P > 0.05). The developed system can be applied to detect other small molecules using appropriate affinity binding pairs.     

The orientationally controlled assembly of genetically modified enzymes in an amperometric biosensor

A novel, nitroreductase (NTR) containing a sequence of six cysteine amino acids, enabling strong thiolate bonds to form on a gold electrode surface without the loss of enzyme activity, was genetically engineered. The enzyme was directly immobilised at a gold electrode without the need for pre-treatment of the surface with a self-assembled monolayer or a conducting polymer. The ensemble was used to develop an amperometric biosensor for the detection of explosives containing nitroaromatic compounds. Preliminary results demonstrate detection levels down to 100 parts per trillion, signifying tremendous promise towards an in situ sensor for the detection of explosives.     

Ultrasensitive electrochemical immunosensor for ochratoxin A using gold colloid-mediated hapten immobilization

A convenient, specific, and highly sensitive electrochemical immunosensor based on an indirect competitive assay format was developed for the determination of ochratoxin A (OTA), a common toxic contaminant in various kinds of agricultural products. The sensing substrate was prepared using a gold electrode modified with a self-assembled monolayer of 1,6-hexanedithiol that mediated the assembly of a gold colloid layer, which could enhance the surface loading of OTA-ovalbumin conjugate and improve the sensitivity in electrochemical readouts. After competition of the limited anti-OTA mouse monoclonal antibody between immobilized hapten and OTA analyte in sample solution, alkaline phosphatase (ALP)-labeled horse anti-mouse immunoglobulin G (IgG) antibody was selectively bound onto the surface of the electrode, affording an indicator for OTA concentration in the sample. Electrochemical response arising from the oxidation of enzymatic product of 1-naphthyl phosphate was observed to be inversely proportional to OTA concentration in the range from 10 pg/ml to 100 ng/ml with a detection limit as low as 8.2 pg/ml. Furthermore, a negligible matrix effect and good recoveries were obtained in the determination of corn samples, evidencing the feasibility of the proposed method for accurate determination of OTA in corn samples.     

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.     

A method for application of samples to matrix-assisted laser desorption ionization time-of-flight targets that enhances peptide detection

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry has become a fundamental tool for the identification and analysis of peptides and proteins. MALDI-TOF is well suited for the analysis of complex biological mixtures because samples are crystallized onto a solid support that can be washed to remove contaminants and salts prior to laser desorption. A number of approaches for immobilizing samples onto MALDI targets have been put forth. These include the use of different chemical matrices and the immobilization of samples onto different solid supports. In large part though, the preparation of MALDI targets has been an empirical exercise that often requires a unique series of conditions for every sample. Here, a simple method for the application of peptide mixtures onto MALDI targets is put forth. This method differs because peptides are added directly to a sample of nitrocellulose dissolved in acetone, allowing them to interact in solution-phase organic solvent. This solution-phase mixture is then spotted to the MALDI target and evaporated, forming a homogenous solid surface for laser desorption. This procedure is robust, highly sensitive, tolerant to detergents, and easily learned. In our hands, the method provides as much as a 10-fold enhancement to the detection of tryptic peptide fragments derived from in-gel digests.