An immunoaffinity tandem mass spectrometry (iMALDI) assay for detection of Francisella tularensis

Francisella tularensis (F. tularensis) has been designated by the CDC as 1 of the 10 organisms most likely to be engineered for bioterrorism. Symptoms of tularemia in humans are non-specific, thus making the disease difficult to diagnose. If not quickly diagnosed and treated, the disease has a high mortality rate - thus methods for early and specific diagnosis are of critical importance.This immunoaffinity MALDI MS/MS (iMALDI) assay provides unambiguous detection of F. tularensis peptides at attomole levels from peptide solutions, and at low CFU levels from bacteria. The addition of stable-labeled versions of the peptide as internal standards allows absolute quantitation of F. tularensis peptides with a linear dynamic range spanning two orders of magnitude. The ability of mass spectrometry to obtain amino acid sequence data on affinity-captured peptides provides absolute specificity and avoids “false positives” from the non-specific binding. The F. tularensis iMALDI assay has been applied to different samples, such as nasal swabs.This novel quantitative diagnostic F. tularensis iMALDI assay allows the safe, sensitive, and specific detection of F. tularensis. The assay can be easily adapted to other target peptides and therefore has broad application potential in clinical diagnosis of other pathogens and diseases.     

Serological Diagnosis of Tularemia in Mice Using the Amperometric Immunosensor

Tularemia caused by the bacterium Francisella tularensis belongs to danger infective diseases, therefore timely diagnosis is an important part of protective activities. A novel approach for serological diagnostics of tularemia using a four‐channel amperometric immunosensor was proposed and evaluated on BALB/c mice as a model organism. The immunosensor was able to work with small volumes of serum samples (0.05 μL per assay) and diagnosed tularemic infection very early after immunization. From the 5^th day, it was possible to distinguish tularemic sera from control sera from mice immunized with Escherichia coli on the probability level of 0.99 (t‐test). Some 40 measurements per one hour can be realized with the developed procedure. The obtained results were confirmed by the standard indirect ELISA. The proposed immunosensor‐based approach seems promising for rapid detection of microbial pathogen infections.     

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10²–10⁵ cfu mL⁻¹ and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor.     

Detection of pathogenic Staphylococcus aureus bacteria by gold based immunosensors

We describe the elaboration of ultra-sensitive immunosensors, to detect the bacterial pathogen Staphylococcus aureus. We utilized commercially available polyclonal anti-S. aureus antibody as receptor. Immunosensors were elaborated by building a self-assembled monolayer (SAM) of thiolamine onto planar gold-coated sensor chips. Then, Protein A was covalently linked to the thiolated SAM using glutaraldehyde as cross-linking agent. After a blocking step by Bovine Serum Albumin (BSA), the antibody was immobilized by affinity to Protein A. This step-by-step construction was monitored by Polarization Modulation Reflection Absorption Infrared Spectroscopy (PM-RAIRS) and Quartz Crystal Microbalance with Dissipation (QCM-D). In a first stage, the parameters of immunosensor elaboration were optimized using a model rabbit IgG. The accessibility of receptors and the homogeneity of their distribution were checked by PM-RAIRS, QCM-D, and by immuno-gold scanning electron microscopy. Then, the specific rabbit anti-S. aureus antibody was immobilized and the resulting sensing layer was applied to the detection of the pathogen target. Independent detection of bacteria immobilized on the sensors by fluorescent imaging allowed validation of the specificity of recognition toward the pathogen as well as a quantitative response of the sensor. Using PM-RAIRS as transducing technique allowed us to enhance sensitivity and reach a very competitive detection level (10⁵ CFU mL^?1).     

Capacitive sensing of microcystin variants of Microcystis aeruginosa using a gold immunoelectrode modified with antibodies,gold nanoparticles and polytyramine

We report on the application of an automated and easy-to-use device to directly measure the immunoreactions between adda-specific monoclonal antibodies and microcystins. The antibodies were immobilized on a gold electrode whose surface was modified first with polytyramine and then with gold nanoparticles. The immunoreaction leads to a change in the capacitance of the system. Under optimum conditions, the sensor is capable of performing stable regeneration-assay cycles and has a low detection limit at a concentration of 0.01 pM level of microcystin-leucine-arginine (MC-LR). The surface of the biosensor can be regenerated with pH 2.5 glycine buffer which dissociates the antibody-antigen complex. The biosensor was used to monitor the production of microcystins during batch cultivation of Microcystis aeruginosa (isolated from ponds in Botswana). Liquid chromatography coupled to MS/MS detection was used to identify three variants, viz. MC-LR (995.6 Da), DmMC-LR (981.2 Da) and MC-LA (910.5 Da).

Photoelectrochemical immunodiagnosis of canine leishmaniasis using cadmium-sulfide-sensitized zinc oxide modified with synthetic peptides

A photoelectrochemical immunosensor for the detection of anti-Leishmania infantum antibodies based on zinc oxide and cadmium sulfide films electrodeposited on an ITO-coated glass slide (CdS/ZnO/ITO) has been proposed. The effects of light/dark conditions and the kinetics of CdS sensitizer regeneration were evaluated by scanning electrochemical microscopy in feedback mode. The platform was modified using two different peptides (PEP 13 and PEP 16) from two different proteins of high specificity and selectivity toward recognition of L. infantum antibodies, producing Peps/CdS/ZnO/ITO. This photoelectrochemical immunosensor provides a cheap and promising method of discriminating between positive and negative canine serum samples.     

A Novel Electrochemical Immunosensor for Prostate-Specific Antigen Based on Noncovalent Nanocomposite of Ferrocene Monocarboxylic Acid with Graphene Oxide

A novel electrochemical immunosensor was developed for the determination of prostate-specific antigen based on immobilization of appropriate antibodies on gold nanoparticles and a poly-(2,6-pyridinediamine) modified electrode. The nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide was prepared by a π-π stacking interaction and was used as the electrochemical probe. A sandwich-type complex immunoassay was applied with polyclonal prostate-specific antigen antibodies labeled with the nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide. In order to improve the sensitivity, a potentiostatic method was used to reduce graphene oxide. Cyclic voltammetry and differential pulse voltammetry were employed to characterize the assembly process and the performance of the immunosensor. Under optimal conditions, the peak current of the immunosensor increased with concentration, showing a linear relationship between the peak current and the logarithm of the prostate-specific antigen concentrations in a wide range of 2.0 pg mL^?1 to 10.0 ng mL^?1 with a low detection limit of 0.5 pg mL^?1. The immunosensor was used for the determination of prostate-specific antigen in serum.     

An electrochemical magneto immunosensor (EMIS) for the determination of paraquat residues in potato samples

An electrochemical magneto immunosensor for the detection of low concentrations of paraquat (PQ) in food samples has been developed and its performance evaluated in a complex sample such as potato extracts. The immunosensor presented uses immunoreagents specifically developed for the recognition of paraquat, a magnetic graphite–epoxy composite (m-GEC) electrode and biofunctionalized magnetic micro-particles (PQ1-BSAMP) that allow reduction of the potential interferences caused by the matrix components. The amperometric signal is provided by an enzymatic probe prepared by covalently linking an enzyme to the specific antibodies (Ab198-cc-HRP). The use of hydroquinone, as mediator, allows recording of the signal at a low potential, which also contributes to reducing the background noise potentially caused by the sample matrix. The immunocomplexes formed on top of the modified MP are easily captured by the m-GEC, which acts simultaneously as transducer. PQ can be detected at concentrations as low as 0.18?±?0.09 μg L^?1. Combined with an efficient extraction procedure, PQ residues can be directly detected and accurately quantified in potato extracts without additional clean-up or purification steps, with a limit of detection (90 % of the maximum signal) of 2.18?±?2.08 μg kg^?1, far below the maximum residue level (20 μg kg^?1) established by the EC. The immunosensor presented here is suitable for on-site analysis. Combined with the use of magnetic racks, multiple samples can be run simultaneously in a reasonable time.     

Sensitive detection of thyroid stimulating hormone by inkjet printed microchip with a double signal amplification strategy

In this paper, we reported a sensitive electrochemical immunosensor coupling protein A/G@magnetic beads and an ALP-based enzymatic-electrochemical reaction on the inkjet printing microchips for the determination of thyroid stimulating hormone.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

Immobilization strategies of Brucella particles on optical fibers for use in chemiluminescence immunosensors

Immunosensors are powerful analytical tools in clinical and veterinary diagnostics. This has led us to design a chemiluminescent immunosensor aimed at identifying anti-Brucella antibodies using optical fibers as the transducer. In order to develop the optimal transducer, to achieve an optimal chemical modification thereby allowing an optimal covalent binding of the protein receptor, several cleaning strategies and silane coupling agents were investigated. Brucella killed organisms were used as a model receptor for quantifying anti-Brucella IgG antibodies in a suspension compared to conventional colorimetric and chemiluminescent ELISA. A silane-benzophenone derivative was selected as the best performing silane coupling agent: the optical fiber immunosensor (OFIS) has showed the lowest limit of detection at 0.207 μg/ml, compared to 0.828 μg/ml and 0.414 μg/ml achieved by colorimetric and chemiluminescent ELISAs, respectively. These results, together with the additional advantages of rapidity, lower reagent volumes and moderate operating conditions, have set the grounds for further study in order to adapt this platform for on-site diagnostics of brucellosis disease markers.     

Multiplexed Detection of Waterborne Pathogens in Circular Microfluidics

Microfluidic lab-on-a-chip presents an ideal solution for bacterial sensing and identification due to its advantages like large surface-to-volume ratio, requirement of low sample volume and multiplexing possibility. The present work deals with the development of an immunosensor chip using circular microchannels fabricated directly with microdimensional copper wire and permanent magnet for capture of Fe₃O₄ magnetic nanoparticle (MNP) conjugate. The MNP facilitate capture of the antigen in a confined space and hence, enhanced fluorescence signal for detection. The multiplexed microfluidic chip permits visual detection and quantification of waterborne pathogens viz. Escherichia coli and Salmonella typhimurium simultaneously. CdTe quantum dots (QDs) with different emission wavelengths were conjugated with anti-E. coli and anti-S. typhimurium antibodies for concurrent fluorescence detection. The present technique provides an inexpensive yet powerful tool to image and quantify pathogens at low numbers with passage of large sample volumes.     

Disposable Immunosensor for Escherichia Coli O157:H7 Based on a Multi-Walled Carbon Nanotube-Sodium Alginate Nanocomposite Film Modified Screen-Printed Carbon Electrode

A disposable immunosensor for the detection of Escherichia coli O157:H7 based on a multiwalled carbon nanotube–sodium alginate nanocomposite film was constructed. The nanocomposite was placed on a screen-printed carbon electrode, and horseradish peroxidase-labeled antibodies were immobilized to E. coli O157:H7 on the modified electrode to construct the immunosensor. The modification procedure was characterized by atomic force microscopy and cyclic voltammetry. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to E. coli O157:H7 in a concentration range of 10³–10¹⁰ cfu/mL, with a relatively low detection limit of 2.94 × 10² cfu/mL (S/N = 3). This immunosensor exhibited satisfactory specificity, reproducibility, stability, and accuracy, making it a potential alternative tool for early assessment of E. coli O157:H7.     

Sandwich-type amperometric immunosensor for human immunoglobulin G using antibody-adsorbed Au/SiO2 nanoparticles

A novel sandwich-type electrochemical immunosensor for human immunoglobulin G (hIgG) was developed using Au/SiO₂ nanoparticles (NPs) with adsorbed horseradish peroxidase-anti-hIgG as the secondary antibody layer. The signal readout is based on the amperometric response to the catalytic reduction of hydrogen peroxide at an AuNPs-polythionine modified glassy carbon electrode. Under optimized conditions, the linear range is from 0.1 to 200 ng·mL^?1, with a detection limit of 0.035 ng·mL^?1 (at an S/N of 3). The immunosensor exhibited a performance that is better than that based on Au/SiO₂NPs-excluded secondary antibody.     

QCM‐Based Immunosensor for the Determination of Ochratoxin A

Abstract

A piezoelectric immunosensor based on a competitive format was developed for determination of ochratoxin A (OTA) concentration. Surface modifications via two self‐assembled monolayers (SAMs) were investigated respectively and a better result was obtained with the SAM of 16‐mercaptohexadecanoic acid (16‐MHDA). The quartz crystal microbalance (QCM)‐based immunosensor was fabricated by immobilizing anti‐OTA antibodies onto the surface of the 16‐MHDA‐modified electrode, and allowing competition between free OTA and that conjugated with BSA to occur. The assay exhibited a working range of 50–1000 ng/mL and a detection limit of 16.1 ng/mL. Studies of interference and matrix effects were performed to evaluate the feasibility of the developed immunosensor for the direct analysis of OTA in real samples. Recoveries were conducted at 50, 200, and 1000 ng/g and were determined to be in the range of 142%–76%. The OTA assay is specific. No cross‐reactivates were observed with citrinin.     

Characterisation of electrochemical immunosensor for detection of viable not-culturable forms of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> in water samples

Legionella pneumophila may cause a fatal pneumonia in humans known as Legionnaires’ disease (LD). The strategies of L. pneumophila to adapt to and resist stressful environmental conditions include the ability to enter into a VBNC (viable but not culturable) state. The detection of L. pneumophila in environmental samples benefits from the use of standardised methods: for detection and enumeration following membrane filtration (AFNOR T90-431, ISO 11731) and detection and quantification by polymerase chain reaction PCR (AFNOR T90-471, ISO 12869). Culture is hampered by its inability to detect VBNC forms and PCR is unable to discriminate between live and dead bacteria. The present immunosensor was obtained by the immobilisation of a monoclonal anti-L. pneumophila antibody (MAb) on an indium-tin oxide (ITO) electrode by the self-assembled monolayers (SAMs) method using an aminosilane. The immunosensor was characterised by wettability (contact angle measurement), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), and electrochemical impedance spectroscopy (EIS). A limit of detection of 10 bacteria per mL was observed on artificial samples.     

An electrochemical immunosensor for the tumor marker α-fetoprotein using a glassy carbon electrode modified with a poly(5-formylindole), single-wall carbon nanotubes,and coated with gold nanoparticles and antibody

We report on a label-free electrochemical immunosensor for α-fetoprotein (α-FP). It is based on the use of a glassy carbon electrode that was first modified with conducting poly(5-formylindole) and single-walled carbon nanotubes (P5FIn/SWNTs), and then coated with gold nanoparticles and the respective antibody. The presence of aldehyde groups warrants direct immobilization of the antibody and results in a convenient method for fabricating of the immunosensor. Gold nanoparticles (GNPs) were deposited on the P5FIn/SWNTs composite material, and the modified electrode was applied to the detection of α-FP. The analytical signal is obtained by measuring the change of amperometric response at a typical working voltage of 100 mV before and after the immunoreaction. The detection limit is 200 fg mL^?1. The immunosensor is simple, sensitive, specific and reproducible. It has the potential for reliable point-of-care diagnosis of tumor or other diseases. Figure

A simple electrochemical immunosensor based on conducting poly(5-formylindole) and single-walled carbon nanotubes composite was fabricated to detect alpha-fetoprotein. The detection limit is 200 fg mL^?1. This immunosensor is simple, sensitive, specific and reproducible.     

A robust sensor platform for label-free detection of anti-Salmonella antibodies using undiluted animal sera

We present a portable and easy-to-use biosensor platform, allowing for label-free detection of diagnostic markers in undiluted animal serum. Exemplarily, this is shown for the detection of anti-Salmonella antibodies. 1-lambda-Reflectometry was used as detection method, making the new biosensor platform portable, cheap, and robust. As recognition elements, lipopolysaccharides (LPSs) from Salmonella typhimurium bacteria were immobilized as sensitive layer on the transducer to carry out serological tests via a direct assay format. For this purpose, a new surface preparation protocol has been worked out allowing for immobilization of the LPS via hydrophobic interactions. It has been shown that results obtained by 1-lambda-Reflectometry are equivalent to those obtained by the non-portable Reflectometric Interference Spectroscopy setup. The new sensor platform was calibrated in both matrices, buffer and undiluted serum. Good sensitivity, selectivity and intra chip reproducibility have been observed. Furthermore, inter chip reproducibility was examined and recovery rates were found to be between 99 and 117 % in undiluted serum.     

Ultrasensitive electrochemiluminescent immunosensor based on dual signal amplification strategy of gold nanoparticles-dotted graphene composites and CdTe quantum dots coated silica nanoparticles

A facile and ultrasensitive electrochemiluminescent (ECL) immunosensor for detection of prostate-specific antigen (PSA) was designed by using CdTe quantum dots coated silica nanoparticles (SiO₂@QDs) as bionanolabels. To construct such an electrochemiluminescence immunosensor, gold nanoparticles-dotted graphene composites were immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies as well as improve the electronic transmission rate. The as-prepared SiO₂@QDs used as bionanolabels, showed good ECL performance and good ability of immobilization for secondary antibodies. The approach provided a good linear response ranging from 0.005 to 10 ng?mL^?1 with a low detection limit of 0.0032 ng?mL^?1. Such immunosensor showed good precision, acceptable stability, and reproducibility. Satisfactory results were obtained for determination of PSA in human serum samples. Therefore, the proposed method provides a new promising platform of clinical immunoassay for other biomolecules.     

A novel immunosensor for carcinoembryonic antigen based on poly(diallyldimethylammonium chloride) protected prussian blue nanoparticles and double-layer nanometer-sized gold particles

A highly sensitive amperometric immunosensor has been developed for the detection of carcinoembryonic antigen (CEA). It is based on (a) Prussian Blue nanoparticles coated with poly(diallyldimethylammonium chloride) (P-PB) and (b) double-layer gold nanocrystals. The sensor was obtained by first electrodepositing porous gold nanocrystals on the glassy carbon electrode (GCE), and then by modifying the electrode with the coated P-PB. Subsequently, colloidal gold nanoparticles (nano-Au) were adsorbed onto the GCE by electrostatic interactions between the negatively charged nano-Au and the positively charged P-PB to immobilize CEA antibodies. Finally, bovine serum albumin was employed to block possible remaining active sites and to prevent the non-specific adsorption on the nano-Au. This immunosensor was characterized by cyclic voltammetry and scanning electron microscopy. The working range was adjusted to two concentration ranges, viz. from 0.5 to 10 ng.mL^?1, and from 10 to 120 ng.mL^?1 of CEA, with a detection limit of 0.2 ng.mL^?1 at three times the background noise.