Immunocomplex Formation Research Articles

Heparin‐gold Nanoparticles and Liquid Crystal Applied in Label‐free Electrochemical Immunosensor for Prostate‐specific Antigen

AbstractA label‐free electrochemical immunosensor based on the liquid crystal (E)‐1‐decyl‐4‐[(4‐decyloxyphenyl)diazenyl]pyridinium bromide (Br−Py), together with heparin‐stabilized gold nanoparticles (AuNP‐Hep) and Nafion is proposed for the determination of prostate‐specific antigen (PSA). The Br−Py liquid crystal presented redox properties and good film‐forming abilities on the electrode surface, and thus it is a suitable alternative as a redox probe for a label‐free electrochemical immunosensor, which could simplify the analysis methodology. The stepwise construction of the immunosensor and the incubation process (immunocomplex formation) were characterized by voltammetry and electrochemical impedance spectroscopy. The proposed immunosensor could directly detect PSA concentrations in the incubation samples, based on the suppression of the Br−Py redox peak (‘base peak’) current. After optimization, the immunosensor exhibited a linear response to PSA concentrations in the range of 0.1 to 50 ng mL−1, with a calculated detection limit of 0.08 ng mL−1. The reproducibility (coefficient of variance less than 3.0 %), selectivity and accuracy of the methodology were adequate. The immunosensor was satisfactorily applied in the quantification of PSA in human blood plasma samples.

TEMPO-based immuno-lateral flow quantitative detection of dengue NS1 protein

The development of a rapid, affordable, and sensitive diagnostic kit for point-of-care is important in most healthcare settings. In this follow-up paper to our previous work on quantification of dengue NS1 protein via impedimetric measurement, our present technology aims to provide quantification by utilizing proprietary stabilized and improved electroactive immunonanoparticles that bind to the target biomarker and subsequently move along toward the biofunctionalized screen-printed gold electrodes (SPGE) to generate an amperometric signal. The SPGE functions simultaneously as a signal transducer and a solid-state support for a sandwich ELISA-like immunoassay. The successful immunocomplex formation is then recorded electrochemically using a potentiostat, whereby the signal was contributed by the presence of a more hydrophilic redox label than ferrocene, namely radical TEMPO (TEMPO), on the formulated nanoparticles. In this paper, a bifunctional ligand, thiolated polyethylene glycol (PEG-thiol) polymer, was used to stabilize 20 nm gold colloidal nanoparticles (AuNPs) in the formulation. PEG was incorporated to not only prevent the salt-mediated AuNPs aggregations, but also provide an anchor for antibody and redox species conjugation. To-date, we have successfully miniaturized a 3D-printed prototype device able to sensitively detect and quantify dengue NS1 protein with only 0.6 μL human clinical serum samples diluted in a volume ratio of 1:100 (PBS diluent) in less than 30 min with a simple cyclic voltammetry analysis. The positive and negative samples were also tested with ELISA, a gold standard validation method, which means that the development of the prototype is a promising start as a point-of-care diagnostic technology.

Immunocontrolling Graphene Oxide Catalytic Nanogold Reaction and Its Application to SERS Quantitative Analysis.

The gold nanoreaction between HAuCl4 and H2O2 is very slow at 50 °C, and the nanoenzyme of graphene oxide (GO) greatly catalyzes the nanoreaction to form gold nanoparticles (AuNPs) with high SERS activity in the presence of Vitoria blue 4R (VB4r) molecular probes, strong resonance Rayleigh scattering (RRS), and surface plasmon resonance (SPR) absorption effect. With the increase of GO, the SERS, RRS, and SPR absorptions were enhanced linearly due to the formation of more AuNPs. The rabit antibody of human chorionic gonadotropin (RHCG) strongly adsorbed on the GO surface to inhibit its catalysis. Upon addition of human chorionic gonadotropin (HCG), the RHCG is separated from the GO surface due to the formation of HCG-RHCG specific immunocomplexes, which led to the recovery of GO catalysis. Using the new strategy of immunocontrolling GO catalysis, three types of resonance methods including SERS, RRS, and surface plasmon resonance (SPR) absorption have been developed for detection of HCG.

Photoluminescent lateral flow based on non-radiative energy transfer for protein detection in human serum

A new paper-based lateral flow immunoassay configuration was engineered and investigated. The assay is intended for the detection of a model protein in human serum, that is, human immunoglobulin G, with the aim to demonstrate a virtually universal protein detection platform. Once the sample is added in the strip, the analyte is selectively captured by antibody-decorated silica beads (Ab-SiO2) onto the conjugate pad and the sample flows by capillarity throughout the strip until reaching the test line, where a sandwich-like immunocomplex takes place due to the presence of antibody-functionalized QDs (Ab-QDs) onto the test line. Eventually, GO is added as a revealing agent and the photoluminescence of those sites protected by the complex Ab-SiO2/Antigen/Ab-QDs will not be quenched, whereas those photoluminescent sites directly exposed are expected to be quenched by GO, including the control line, made of bare QDs, reporting that the assay occurred successfully. Hence, the photoluminescence of the test line is modulated by the formation of sandwich-like immunocomplexes. The proposed device achieves a limit of detection (LOD) of 1.35ngmL−1 in standard buffer, which is lower when compared with conventional lateral flow technology reported by gold nanoparticles, including other amplification strategies. Moreover, the resulting device was proven useful in human serum analysis, achieving a LOD of 6.30ngmL−1 in this complex matrix. This low-cost disposable and easy-to-use device will prove valuable for portable and automated diagnostics applications, and can be easily transferred to other analytes such as clinically relevant protein biomarkers.

Straightforward Immunosensing Platform Based on Graphene Oxide‐Decorated Nanopaper: A Highly Sensitive and Fast Biosensing Approach

Immunoassays are nowadays a crucial tool for diagnostics and drug development. However, they often involve time‐consuming procedures and need at least two antibodies in charge of the capture and detection processes, respectively. This study reports a nanocomposite based on graphene oxide‐coated nanopaper (GONAP) facilitating an advantageous immunosensing platform using a single antibody and without the need for washing steps. The hydrophilic, porous, and photoluminescence‐quenching character of GONAP allows for the adsorption and quenching of photoluminescent quantum dots nanocrystals complexed with antibodies (Ab‐QDs), enabling a ready‐to‐use immunosensing platform. The photoluminescence is recovered upon immunocomplex (antibody‐antigen) formation which embraces a series of interactions (hydrogen bonding, electrostatic, hydrophobic, and Van der Waals interactions) that trigger desorption of the antigen‐Ab‐QD complex from GONAP surface. However, the antigen is then attached onto the GONAP surface by electrostatic interactions leading to a spacer (greater than ≈20 nm) between Ab‐QDs and GONAP and thus hindering nonradiative energy transfer. It is demonstrated that this simple—yet highly sensitive—platform represents a virtually universal immunosensing approach by using small‐sized and big‐sized targets as model analytes, those are, human‐IgG protein and Escherichia coli bacteria. In addition, the assay is proved effective in real matrices analysis, including human serum, poultry meat, and river water. GONAP opens the way to conceptually new paper‐based devices for immunosensing, which are amenable to point of care applications and automated diagnostics.

A sensitive electrochemical immunosensor based on poly(2-aminobenzylamine) film modified screen-printed carbon electrode for label-free detection of human immunoglobulin G

This work focuses on fabricating poly(2-aminobenzylamine)-modified screen-printed carbon electrode as an electrochemical immunosensor for the label-free detection of human immunoglobulin G. To selectively detect immunoglobulin G, the anti-immunoglobulin G antibody with high affinity to immunoglobulin G was covalently linked with the amine group of poly(2-aminobenzylamine) film-deposited screen-printed carbon electrode. The selectivity for immunoglobulin G was subsequently assured by being challenged with redox-active interferences and adventitious adsorption did not significantly interfere the analyte signal. To obviate the use of costly secondary antibody, the [Fe(CN)6]4-/3- redox probe was instead applied to measure the number of human immunoglobulin G through the immunocomplex formation that is quantitatively related to the level of the differential pulse voltammetric current. The resulting immunosensor exhibited good sensitivity with the detection limit of 0.15 ng mL−1, limit of quantitation of 0.50 ng mL−1 and the linear range from 1.0 to 50 ng mL−1. Given those striking analytical performances and the affordability arising from using cheap screen-printed carbon electrode with label-free detection, the immunosensor serves as a promising model for the next-step development of a diagnostic tool.

Signal-On Photoelectrochemical Immunoassay for Aflatoxin B1 Based on Enzymatic Product-Etching MnO2 Nanosheets for Dissociation of Carbon Dots.

Aflatoxin B1 (AFB1) monitoring has attracted extensive attention because food safety is a worldwide public health problem. Herein, we design a novel simultaneously visual and photoelectrochemical (PEC) immunosensing system for rapid sensitive detection of AFB1 in foodstuff. The immunoreaction was carried out on anti-AFB1 antibody-modified magnetic beads by using glucose oxidase (GOx)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugates as the tags with a competitive-type immunoassay format, while the visual and PEC evaluation was performed via carbon quantum dots (CQDs)-functionalized MnO2 nanosheets. Accompanying the formation of immunocomplexes, the carried GOx initially oxidized the substrate (glucose) for the generation of H2O2, which reduced/etched MnO2 nanosheets into Mn2+ ions, thereby resulting in the dissociation of CQDs from the electrode. Within the applied potentials, the photocurrent of MnO2-CQDs-modified electrode decreased with the increasing H2O2 level in the detection cell. Meanwhile, a visual detection could be performed according to the change in the color of MnO2-CQDs-coated electrode. To elaborate, this system was aggregated into a high-throughput microfluidic device to construct a semiautomatic detection cell. Under optimal conditions, the photocurrent increased with the increasing target AFB1 within a dynamic working range from 0.01 to 20 ng mL-1 with a limit of detection (LOD) of 2.1 pg mL-1 (ppt). The developed immunoassay exhibited good reproducibility and acceptable accuracy. In addition, the method accuracy relative to AFB1 ELISA kit was evaluated for analyzing naturally contaminated or spiked peanut samples, giving the well-matched results between two methods. Although our strategy was focused on the detection of target AFB1, it is easily extended to screen other small molecules or mycotoxins, thereby representing a versatile immunosensing scheme.

Nanomaterial Labels for Signal Amplification in Electrochemical Immunosensors

Electrochemical immunosensors are functions by measuring the changes in the electrical properties such as current, potential, conductance, capacitance, and impedance during an immunochemical reaction between antigen (target molecule) and the antibody(Pandiaraj et al., 2014; Vasudev et al., 2013). Although capacitive and impedimetric immunosensors can directly be utilized to investigate the antibody-antigen interaction without the need of other reagents and separation steps, their analytical sensitivity is limited in clinical applications. Amperometric immunosensors are based on the measurement of currents resulting from the electrochemical oxidation or reduction of electroactive species had attracted more interests recently for a higher sensitivity and less complicated instrumentation (Rusling et al., 2010). However, amperometric immunosensors requires labelling of either antigen or antibody, since both the reaction partners are electrochemically inert. Labelling is a time consuming process, costly and denatures the biomolecules. Amperometric immunosensors that utilizes electrolyte solution containing reversible redox species, such as ferrocene and its derivatives, dyes, hydroquinone, hexacyanoferrates etc. have been widely used as mediators or signal generating probes instead of labelling antibodies/antigens; however this would cause electrode contamination, reagent-consumption and operation inconvenience. To solve these problems, it should be preferable to immobilize the signal generating probe on electrode surfaces to construct label-free amperometric immunosensors. The use of nanomaterials as signal generating probes for electrochemical detection of biomarkers have been reviewed earlier (Liu and Lin, 2007). However, the selection of signal generating probe is very important for electrochemical immunosensor, especially for label-free electrochemical immunosensor, since it is responsible for three major tasks: (i) being redox active and produce electrochemical signals for corresponding immunocomplex formation, (ii) increasing specific surface area of electrode to immobilize more antibodies and improving conductivity for sensitive detection of antigen. For electrochemical devices, nanoparticles, carbon nanotubes and graphene have been used to catalyze electrochemical reactions and to improve electron transfer. Nanoparticles, especially have also been used as labels to generate intense signal in comparison with electroactive molecules or organometallic complexes like ferrocene. Indeed, due to their high number of atoms, huge number of electron can be exchanged through oxidation or reduction. Group 11 metals (Cu, Ag, and Au), which are very weak chemisorbers, often display marked catalytic and electrocatalytic properties. Some types of copper electrodes showed an excellent electrocatalytic activity for DNA detection without observable self-poisoning. Copper electrodes undergo oxidation at unexpectedly low potentials, typically within the so-called double layer region, to form surface-bonded copper(I) hydrous oxide species. A reagentless, label-free voltammetric immunosensing strategy was demonstrated by employing nanomaterial integrated electrode as a signal generating probe. Copper/copper oxide (Cu/CuxO) nanoparticles integrated screen printed carbon electrodes (SPCE) were used to construct the immunosensing platform. The stress biomarker, cortisol was employed as a model analyte. Cortisol specific monoclonal antibodies (C-Mab) were covalently immobilized on the surface of the Cu/CuxO-SPCE functionalized with dithiobis(succinimidyl propionate) (DTSP) self-assembled monolayer (SAM). The formation of Cu/CuxO nanoparticles on the surface of SPCE was characterized by scanning electron microscopy (SEM), and X-ray diffraction (XDR) analysis. The redox signal of the Cu/CuxO nanoparticles could be detected directly through cyclic voltammetry and used as signal generation probe for the direct detection of cortisol. The formation of immunocomplexes leads to a decrease in the electrochemical redox signal of Cu/CuxO nanoparticles owing to increased spatial blocking of the electrode surface. The proposed immunosensing strategy allows a rapid and sensitive means of cortisol analysis with a limit of detection of about 1 pg/mL. The Cu/CuxO platform can be further applied for designing other label-free immunoassays.

B7-H3 Augments Inflammatory Responses and Exacerbates Brain Damage via Amplifying NF-κB p65 and MAPK p38 Activation during Experimental Pneumococcal Meningitis.

The costimulatory protein B7-H3 has been shown to play a contributory role in the development and progression of experimental pneumococcal meningitis by augmentation of the innate immunity-associated inflammatory response via a TLR2-dependent manner. This study aimed to clarify the component(s) of TLR2-mediated signal transduction pathways responsible for B7-H3-augmented inflammatory response and subsequent brain damage during experimental pneumococcal meningitis. Administration of B7-H3 did not augment expression of TLR2 and other TLR2 upstream components, but led to an enhanced formation of MyD88-IRAK immunocomplex in the brain of S. pneumoniae-infected mice. Furthermore, B7-H3 substantially augmented S. pneumoniae-induced activation of TLR2 downstream NF-κB p65 and MAPK p38 pathways in the brain of S. pneumoniae-infected mice. Notably, blockage of NF-κB p65 and/or MAPK p38 with their specific inhibitors strongly attenuated B7-H3-amplified inflammatory response with significantly reduced proinflammatory cytokine and chemokine production, and markedly ameliorated B7-H3-exacerbated disruption of blood-brain barrier and severity of disease status in S. pneumoniae-infected mice. These results indicate that targeting NF-κB p65 and/or MAPK p38 may represent a promising therapeutic option for amelioration of overwhelming inflammatory response-associated brain injury frequently observed during pneumococcal meningitis.

Development of a single-step immunoassay microdevice based on a graphene oxide-containing hydrogel possessing fluorescence quenching and size separation functions.

An immunoassay, which is an indispensable analytical method both in biological research and in medical fields was successfully integrated into a "single-step" by developing a microdevice composed of a graphene oxide (GO)-containing hydrogel and a poly (dimethylsiloxane) (PDMS) microchannel array with a polyethylene glycol (PEG) coating containing a fluorescently-labelled antibody. Here we used 2-hydroxyethylmethacrylate (HEMA) as a monomer that is easily, and homogeneously, mixed with GO to synthesize the hydrogel. The fluorescence quenching and size separation functions were then optimized by controlling the ratios of HEMA and GO. Free fluorescently-labelled antibody was successfully separated from the immunoreaction mixture by the hydrogel network structure, and the fluorescence was subsequently quenched by GO. In comparison to the previously reported immunoassay system using GO, the present system achieved a very high fluorescence resonance energy transfer (FRET) efficiency (∼90%), due to the use of direct adsorption of the fluorescently-labelled antibody to the GO surface; in contrast, the former reported method relied on indirect adsorption of the fluorescently-labelled antibody via immunocomplex formation at the GO surface. Finally, the single-step immunoassay microdevice was made by combining the developed hydrogel and the PDMS microchannel with a coating containing the fluorescently-labelled antibody, and successfully applied for the single-step analysis of IgM levels in diluted human serum by simple introduction of the sample via capillary action.

Determination of Staphylococcus aureus B-1266 by an Enzyme-Free Electrochemical Immunosensor Incorporating Magnetite Nanoparticles

ABSTRACTA simple and inexpensive immunosensor is reported for the rapid determination of Staphylococcus aureus B-1266 that uses Fe3O4–SiO2–NH2 nanoparticles as the direct signal label. The electrochemical immunoassay procedure includes the incubation of bacteria with excess magnetite nanoparticles, the magnetic separation of the free nanoparticles, a labeled immunocomplex formation on the surface of a planar electrode, and the electrochemical response from the magnetite nanoparticles in the immunocomplex. The electrochemical immunosensor allows for the selective and accurate detection of S. aureus from 10 to 105 CFU mL−1 with a relative standard deviation lower than 10%. The limit of detection was 8.7 CFU mL−1.

Functionalized polymeric magnetic nanoconstructs for selective capturing and sensitive detection of Salmonella typhimurium

Rapid detection and enumeration of pathogens is essential for monitoring contamination and spoilage of food products to ensure improved quality control management. Functionalized polymeric magnetic nanoconstructs (FPMNCs) were developed as an effective immunomagnetic separator and sensing platform for the selective capturing of Salmonella typhimurium. Novel FPMNCs were prepared in three stages involving synthesis of iron oxide (IO) dispersion, capping with sodium oleate and encapsulation of preformed IO nanoparticles by in-situ free radical emulsion polymerization of styrene (St), methyl methacrylate (MMA) and acetoacetoxy ethylmethacrylate (AAEM). PMMA improves the stability of FPMNCs by bridging extremely hydrophobic PS and hydrophilic PAAEM. Core-shell morphology of hydrophobic core of IO, PS & PMMA and hydrophilic shell of PAAEM was demonstrated by SEM, TEM and FTIR studies. FPMNCs with surface functionalized acetoacetoxy groups were covalently attached with polyclonal antibodies against Salmonella common structural antigen (CSA-1-Ab) without using any linker and catalyst. Colorimetric readout signal was acquired using CSA-1-Ab-HRP as secondary antibody after formation of sandwich immunocomplex with bacteria where the optical density of the samples were recorded using ELISA plate reader at 450 nm. The developed immunoassay was specific and selective which captures only targeted S. typhimurium with a detection limit of 10 cells/mL lower than infectious dose of salmonellosis infection. Minimal interference of food matrix with high signal to noise ratio was shown by various food samples. In addition, the performance of developed FPMNC based immunoassay was superior to commercially available immunomagnetic microbeads demonstrating undisputed advantage for capturing and detecting specific bacteria without any pre-enrichment of sample.

A Label-Free Microelectrode Array Based on One-Step Synthesis of Chitosan–Multi-Walled Carbon Nanotube–Thionine for Ultrasensitive Detection of Carcinoembryonic Antigen

Carcinoembryonic antigen (CEA) has been an extensively used tumor marker responsible for clinical early diagnosis of cervical carcinomas, and pancreatic, colorectal, gastric and lung cancer. Combined with micro-electro mechanical system (MEMS) technology, it is important to develop a novel immune microelectrode array (MEA) not only for rapid analysis of serum samples, but also for cell detection in vitro and in vivo. In this work, we depict a simple approach to modify chitosan–multi-walled carbon nanotubes–thionine (CS–MWCNTs–THI) hybrid film through one-step electrochemical deposition and the CS-MWCNTs-THI hybrid films are successfully employed to immobilize anti-CEA for fabricating simple, label-free, and highly sensitive electro-chemical immune MEAs. The detection principle of immune MEA was based on the fact that the increasing formation of the antigen-antibody immunocomplex resulted in the decreased response currents and the relationship between the current reductions with the corresponding CEA concentrations was directly proportional. Experimental results indicated that the label-free MEA had good selectivity and the limit of detection for CEA is 0.5 pg/mL signal to noise ratio (SNR) = 3. A linear calibration plot for the detection of CEA was obtained in a wide concentration range from 1 pg/mL to 100 ng/mL (r = 0.996). This novel MEA has potential applications for detecting CEA for the research on cancer cells and cancer tissue slices as well as for effective early diagnosis.

Visible-light driven photoelectrochemical immunosensor for insulin detection based on MWCNTs@SnS2@CdS nanocomposites

In this work, a label-free photoelectrochemical (PEC) immunosensor was developed for ultrasensitive detection of insulin based on MWCNTs@SnS2@CdS nanocomposites. As graphene-like 2D nanomaterial, SnS2 nanosheets loaded on the conducting framework of multi-walled carbon nanotubes (MWCNTs) were adopted for the construction of immunosensor for the first time, providing a favorable substrate for in-situ growth of CdS nanocrystal that had suitable band structure matching well with SnS2. The well-matched band structure of these two metal sulfides effectively inhibited the recombination of photogenerated electron-hole pairs, thus improving the photo-to-current conversion efficiency. Besides, the introduction of MWCNTs facilitated electron transfer across the surface of electrodes, leading to a further increment of photocurrent. The as constructed label-free PEC immunosensor based on MWCNTs@SnS2@CdS nanocomposites exhibited excellent PEC performance for the detection of insulin. The concentrations of insulin could be directly detected based on the decrement of photocurrent that was brought by the increased steric hindrances due to the formation of antigen-antibody immunocomplexes. Under the optimal conditions, the PEC immunosensor had a sensitive response to insulin in a linear range of 0.1pgmL−1 to 5ngmL−1 with a detection limit of 0.03pgmL−1. Meanwhile, good stability and selectivity were achieved as well. The design and fabrication of this PEC immunosensor based on MWCNTs@SnS2@CdS nanocomposites not only provided an ideal platform for the detection of insulin, but also opened up a new avenue for the development of immunosensor for some other biomarkers analysis.

Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.

Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of β2 and β3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and β sheet fragments make the hCC structure rigid and unable to undergo the swapping process.

A label free electrochemical immunosensor for sensitive detection of porcine serum albumin as a marker for pork adulteration in raw meat

A label free electrochemical immunosensor for sensitive detection of porcine serum albumin (PSA) is reported in this work. The immunosensor was constructed by first electrochemically reducing 4-carboxyphenyl diazonium salt, which had been electrochemically generated in situ, to a stable 4-carboxyphenyl layer on carbon nanofiber-modified screen printed electrode. Antibodies were covalently attached onto the electrode using carbodiimide chemistry between the carboxylic groups of the 4-carboxyphenyl layer and amine groups of the antibody. Using the strong affinities of serum albumins towards anions, the increase in cathodic peak current in anionic redox probe after immunocomplex formation with antibodies was used for PSA detection. The reported immunosensor demonstrated a linear range from 0.5 to 500pg/mL for the measurement of PSA with detection limit of 0.5pg/mL in buffer solution. Cross-reactivity studies have shown excellent specificity with satisfactory recovery of PSA in fresh meat samples without the need of sample dilution.

Photoluminescence detection of 2,4,6-trinitrotoluene (TNT) binding on diatom frustule biosilica functionalized with an anti-TNT monoclonal antibody fragment

A selective and label-free biosensor for detection of the explosive compound 2,4,6-trinitrotoluene (TNT) in aqueous solution was developed based on the principle of photoluminescence quenching of upon immunocomplex formation with antibody-functionalized diatom frustule biosilica. The diatom frustule is an intricately nanostructured, highly porous biogenic silica material derived from the shells of microscopic algae called diatoms. This material emits strong visible blue photoluminescence (PL) upon UV excitation. PL-active frustule biosilica was isolated from cultured cells of the marine diatom Pinnularia sp. and functionalized with a single chain variable fragment (scFv) derived from an anti-TNT monoclonal antibody. When TNT was bound to the anti-TNT scFv-functionalized diatom frustule biosilica, the PL emission from the biosilica was partially quenched due to the electrophilic nature of the nitro (-NO2) groups on the TNT molecule. The dose-response curve for immunocomplex formation of TNT on the scFv-functionalized diatom frustule biosilica had a half-saturation binding constant of 6.4±2.4·10−8M and statistically-significant measured detection limit of 3.5·10−8M. The binding and detection were selective for TNT and TNB (trinitrobenzene) but not RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) or 2,6-DNT (2,6-dinitrotoluene).

Electrochemical lateral flow immunosensor for detection and quantification of dengue NS1 protein.

An Electrochemical Lateral Flow Immunosensor (ELFI) is developed combining screen-printed gold electrodes (SPGE) enabling quantification together with the convenience of a lateral flow test strip. A cellulose glassy fiber paper conjugate pad retains the marker immunoelectroactive nanobeads which will bind to the target analyte of interest. The specific immunorecognition event continues to occur along the lateral flow bed until reaching the SPGE-capture antibodies at the end of the cellulosic lateral flow strip. The rationale of the immunoassay consists in the analyte antigen NS1 protein being captured selectively and specifically by the dengue NS1 antibody conjugated onto the immunonanobeads thus forming an immunocomplex. With the aid of a running buffer, the immunocomplexes flow and reach the immuno-conjugated electrode surface and form specific sandwich-type detection due to specific, molecular recognition, while unbound beads move along past the electrodes. The successful sandwich immunocomplex formation is then recorded electrochemically. Specific detection of NS1 is translated into an electrochemical signal contributed by a redox label present on the bead-immobilized detection dengue NS1 antibody while a proportional increase of faradic current is observed with increase in analyte NS1 protein concentration. The first generation ELFI prototype is simply assembled in a cassette and successfully demonstrates wide linear range over a concentration range of 1-25 ng/mL with an ultrasensitive detection limit of 0.5 ng/mL for the qualitative and quantitative detection of analyte dengue NS1 protein.

Ultrasensitive and accelerated detection of ciguatoxin by capillary electrophoresis via on-line sandwich immunoassay with rotating magnetic field and nanoparticles signal enhancement

A sensitive and rapid on-line immunoassay for the determination of ciguatoxin CTX3C was developed based on a capillary mixing system, which was integrated with capillary electrophoresis (CE) separation and electrochemical (EC) detection. In the sandwich immunoassay system, anti-CTX3C-functionalized magnetic nanoparticles were used as immunosensing probes, and horseradish peroxidase (HRP) and anti-CTX3C antibody were bound onto the surface of gold nanoparticles (AuNPs) and used as recognition elements. Online formation of immunocomplex was realized in capillary inlet end with an external rotating magnetic field. Compared with classical HPLC-MS and ELISA, the assay adopting AuNPs as multienzyme carriers and online sandwich immunoassay format with rotating magnetic field exhibited higher sensitivity and shorter assay time. The linear range of the assay for CTX3C was from 0.6 to 150 ng/L with a correlation coefficient of 0.9948 (n = 2), and the detection limit (S/N = 3) was 0.09 ng/L. The developed assay showed satisfying reproducibility and stability, and it was successfully applied for the quantification of CTX3C in fish samples.

Dose–response curve of a microfluidic magnetic bead-based surface coverage sandwich assay

Magnetic micro- and nanoparticles ('magnetic beads') have been used to advantage in many microfluidic devices for sensitive antigen (Ag) detection. Today, assays that use as read-out of the signal the number count of immobilized beads on a surface for quantification of a sample's analyte concentration have been among the most sensitive and have allowed protein detection lower than the fgmL(-1) concentration range. Recently, we have proposed in this category a magnetic bead surface coverage assay (Tekin et al., 2013 [1]), in which 'large' (2.8μm) antibody (Ab)-functionalized magnetic beads captured their Ag from a serum and these Ag-carrying beads were subsequently exposed to a surface pattern of fixed 'small' (1.0μm) Ab-coated magnetic beads. When the system was exposed to a magnetic induction field, the magnet dipole attractive interactions between the two bead types were used as a handle to approach both bead surfaces and assist with Ag-Ab immunocomplex formation, while unspecific binding (in absence of an Ag) of a large bead was reduced by exploiting viscous drag flow. The dose-response curve of this type of assay had two remarkable features: (i) its ability to detect an output signal (i.e. bead number count) for very low Ag concentrations, and (ii) an output signal of the assay that was non-linear with respect to Ag concentration. We explain here the observed dose-response curves and show that the type of interactions and the concept of our assay are in favour of detecting the lowest analyte concentrations (where typically either zero or one Ag is carried per large bead), while higher concentrations are less efficiently detected. We propose a random walk process for the Ag-carrying bead over the magnetic landscape of small beads and this model description explains the enhanced overall capture probability of this assay and its particular non-linear dose response curves.