Dissociation Rate Kinetics in a Solid-Phase Flow Immunoassay

Abstract

Solid-phase displacement assays allow extremely fast analyses when performed under continuous flow conditions. Continuous dissociation of labeled antigen from the immobilized saturated antibodies occurs even in the absence of competing unlabeled antigen. This spontaneous dissociation creates more unoccupied antibody binding sites which affect the magnitude of the signal generated. In order to evaluate the impact of this phenomenon in more detail, we extended the law of mass action to solid-phase binding assays and analyzed the dissociation kinetics of labeled antigen under continuous flow conditions. The effect of the flow on the dissociation kinetics was determined by calculation of the apparent dissociation rate constants (k_d) which increase with an increase in the flow rate. These dissociation rate constants are approximately 20- to 30-fold lower than those obtained from displacement studies (i.e., in the presence of competing unlabeled antigen). The difference in the dissociation rate constants obtained in the two studies is most likely a function of the degree of reassociation. The results of this study provide a basis for better understanding antibody kinetics at solid-liquid interfaces under flow conditions.     

Surface Plasmon Resonance Immunoassay for Ochratoxin A Based on Nanogold Hollow Balls with Dendritic Surface

Abstract

A surface plasmon resonance (SPR)‐immunosensor based on nano‐size gold hollow ball (GHB) with dendritic surface has been developed for detection of Ochratoxin A (OTA). A thionine thin film was initially electropolymerized onto the SPR‐probe surface, and then anti‐OTA monoclonal antibody (anti‐OTA) was immobilized onto the SPR‐probe surface by means of GHB conjugation. The binding of target molecules onto the immobilized antibodies causes an increase in the resonant angle of the sensor chip, and the resonant angle shift was proportional to the OTA concentration in the range of 0.05–7.5 ng/ml with a detection limit of 0.01 ng/ml at a signal/noise ration of 3. A glycine‐HCl solution (pH 2.8) was used to release antigen‐antibody complexes from the biorecognition surface. Good reusability was exhibited. Moreover, spiking various levels of OTA into three milk samples was assayed using the proposed immunoassay. Analytical results show the precision of the developed immunoassay is acceptable. Compared with the conventional enzyme‐linked immunosorbent assay, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

Extraction,amplification and detection of DNA in microfluidic chip-based assays

This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references. Figure

A graphical presentation of main PCR assays: DNA extraction from raw sample, target amplification by PCR and final product detection in conventional bench-top lab and miniaturized microfluidic chip.     

A Platinum(II) Phenylphenanthroimidazole with an Extended Side‐Chain Exhibits Slow Dissociation from a c‐Kit G‐Quadruplex Motif

A series of three platinum(II) phenanthroimidazoles each containing a protonable side‐chain appended from the phenyl moiety through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) were evaluated for their capacities to bind to human telomere, c‐Myc, and c‐Kit derived G‐quadruplexes. The side‐chain has been optimized to enable a multivalent binding mode to G‐quadruplex motifs, which would potentially result in selective targeting. Molecular modeling, high‐throughput fluorescence intercalator displacement (HT‐FID) assays, and surface plasmon resonance (SPR) studies demonstrate that complex 2 exhibits significantly slower dissociation rates compared to platinum phenanthroimidazoles without side‐chains and other reported G‐quadruplex binders. Complex 2 showed little cytotoxicity in HeLa and A172 cancer cell lines, consistent with the fact that it does not follow a telomere‐targeting pathway. Preliminary mRNA analysis shows that 2 specifically interacts with the ckit promoter region. Overall, this study validates 2 as a useful molecular probe for c‐Kit related cancer pathways.     

Analysis of Neutral Sugars of Asparagus officinalis Linn. Polysaccharide by CZE with Amperometric Detection

Analysis of the neutral sugars of Asparagus officinalis Linn. polysaccharide by different methods has yielded inconsistent results. In the work discussed in this paper, capillary zone electrophoresis with amperometric detection (CZE–AD) was used for analysis of neutral sugars in A. officinalis Linn. polysaccharide. The configuration of the wall-jet and the diameter of the copper disk electrode were investigated to achieve optimized detection sensitivity. The separation electrolyte, separation voltage, and injection time were studied for their effects on CZE separation. Under the optimum CZE–AD conditions, seven monosaccharides were separated to baseline by using 120 mM NaOH as separation electrolyte. Linear response was excellent and repeatability was satisfactory. It was found that Asparagus polysaccharide was composed of fucose, galactose, glucose, rhamnose, arabinose, fructose, and xylose at a mole ratio of 0.2:16.2:5.0:1.0:15.5:0.6:18.8. Compared with other methods, analysis of the composition of Asparagus polysaccharide by CZE–AD had the merits of rapidness, accuracy, and lower sampling volume.

     

Separation of Lactoferrin from Human Saliva Using Monolithic Disc

In this study, we optimized method for the isolation and detection of lactoferrin from human saliva using 3 mm short monolithic disc. We optimized the conditions for separation as flow rate 4 mL min⁻¹ and ionic strength of effluent as 2 M·NaCl. We estimated limit of detection of whole method, which was hyphenated to the Bradford’s assay, down to 100 ng mL⁻¹. The purity of the isolated fractions was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and recovery of isolation was found to be 51 % using minimally processed sample of saliva. Further, we tested the optimized method on group of healthy volunteers (n = 7). We were able to distinguish between the healthy subjects and subject suffering from celiac disease, which reported at least 2.5× higher level of lactoferrin in comparison to healthy ones. The results were correlated with standard enzyme-linked immunosorbent assay (ELISA) kit with obtained correlation coefficient R ² = 0.8446. Analysis of lactoferrin in saliva by monolithic disc and subsequent offline photometric detection is faster and cheaper method compared to ELISA commercial kit. The total analysis of one sample takes <20 min.

     

The Detection of Pseudomonas aeruginosa Using the Quartz Crystal Microbalance

Abstract

The development of a piezoelectric immunosensor for the detection of Pseudomonas aeruginosa in milk and dairy samples was undertaken here. This was achieved primarily by optimising the system using ELISA, investigating capture, competitive and displacement assays. Results from ELISA supplied information on detection limits and linear ranges obtained with each assay. A displacement assay was chosen to be transferred to the piezoelectric system and the reduction in mass on the surface of the crystal due to antigen displacement was measured by recording the frequency changes of the quartz crystal microbalance. The linear range obtained was from 2x10⁶ cell/ml to 1x10⁸ cell/ml and the limit of detection was 100,000 cells. The system was also tested for cross reactivity with a non-specific antigen, Pseudomonas fluorescens.     

Surface plasmon resonance application in prostate cancer biomarker research

Prostate cancer (PCa) diagnostics can be effectively addressed using sensor-based approaches. Proper selection of biomarkers to be included in biosensors for accurate detection becomes the need of the hour. Such biosensor and biochip technologies enable fast and efficient determination of proteins and provide a remarkable insight into the changes in the protein structure, such as aberrant glycosylation, which can increase the performance, sensitivity and specificity of clinic assays. However, for a thorough comprehension of such complex protein modifications, it is crucial to understand their biospecific interactions. Surface plasmon resonance (SPR), one of the most rapidly developing techniques for measuring real-time quantitative binding affinities and kinetics of the interactions of antigens and antibodies, was chosen as an appropriate tool for this purpose. Herein, experiments on the interactions of antibodies specific against different epitopes of free and complexed prostate-specific antigen (PSA), a prominent PCa biomarker, are presented with two main aims: (i) to continue as lectin glycoprofiling studies and; (ii) to be used in microfluidic immunoassay-based platforms for point-of-care devices. Various PSA-specific antibodies were covalently immobilized on the biochip surface via amine coupling, and free or complexed PSA was injected into the dual-flow channels of the SPR device. Kinetic parameters and affinity constants of these interactions, as well as cross-reactivities of the used antibodies were determined. The sandwich assay for PSA determination was developed employing both primary and secondary anti-PSA antibodies. Sensitivity of the assay was 3.63 nM^?1, the detection limit was 0.27 nM and the SPR biosensor response towards free PSA was linear up to 25 nM. All these findings are essential for proper design of a selective, sensitive, and highly reliable biosensor for PCa diagnosis as a lab-on-chip device.     

Time-Resolved Fluorescence Receptor Assay for Benzodiazepines

Abstract

A novel non-isotopic receptor binding technique for the detection of benzodiazepines is described. A benzodiazepine labeled with europium chelate was prepared and employed as a labeled ligand, and time-resolved measurements of the long lifetime fluorescence of europium chelate allowed avoidance of interference due to proteins in the receptor preparation. Experimental results demonstrate a sigmoid inhibition curve, with binding of the labeled ligand inhibited by comparable concentrations of the unlabeled drug. The proposed assay may provide a simple procedure for the measurement of benzodiazepines in biological systems and a useful tool in the screening of natural substances for new classes of benzodiazepine-like compounds.     

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen–antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein–protein interactions under identical conditions. Antibody–antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein–protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules. Figure Microchannel SPR imaging for protein–protein interactions     

Parameters Governing the Formation of Photopolymerized Silica Sol–Gel Monoliths in PDMS Microfluidic Chips

Although polydimethylsiloxane (PDMS) microfluidic chips provide an alternative to more expensive microfabricated glass chips, formation of monolithic stationary phases in PDMS is not a trivial task. Photopolymerized silica sol–gel monoliths were fabricated in PDMS-based microfluidic devices using 3-trimethoxysilylpropylmethacrylate and glycidyloxypropyltrimethoxysilane. The monolith formation was optimized by identifying a suitable porogen, controlling monomer concentration, functional additives, salts, porogen, wall attachment methods, and rinsing procedures. The resulting monoliths were evaluated using scanning electron microscopy, image analysis, differential scanning calorimetry, and separation performance. Monoliths functionalized with boronic acid ligands were used for the separation of cis-diol containing compounds both in batch mode and in the microfluidic chip.

     

Aptamer based surface plasmon resonance sensor for aflatoxin B1

The authors describe a surface plasmon resonance (SPR) based aptasensor for the carcinogenic mycotoxin aflatoxin B1 (AFB1) in a direct assay format. The aptamer is immobilized on the surface of a commercial sensor chip, and the SPR signal increases on binding of AFB1. The sensor chip can be fully regenerated by passing a flow of buffer over it upon which bound AFB1 dissociates from the aptamer. The biosensor works in the 0.4 nM to 200 nM AFB1 concentration range and has a 0.4 nM detection limit. It allows AFB1 to be determined in complex samples such as diluted red wine and beer. The assay is sensitive, and the chip is easily regenerated and stable. The method therefore overcomes certain limitations of antibody-based SPR assays and of competitive SPR assays for AFB1.

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Graphical abstract Schematic presentation of the assay: Aptamer is coated on the chip of SPR, and the binding between aflatoxin B1 (AFB1) and the aptamer on chip causes SPR responses, allowing sensitive detection of AFB1.

     

Rapid capacitive detection of femtomolar levels of bisphenol A using an aptamer-modified disposable microelectrode array

A label-free and single-step method is reported for rapid and highly sensitive detection of bisphenol A (BPA) in aqueous samples. It utilizes an aptamer acting as a probe molecule immobilized on a commercially available array of interdigitated aluminum microelectrodes. BPA was quantified by measuring the interfacial capacitance change rate caused by the specific binding between bisphenol A and the immobilized aptamer. The AC signal also induces an AC electrokinetic effect to generate microfluidic motion for enhanced binding. The capacitive aptasensor achieves a limit of detection as low as 10 fM(2.8 fg ⋅ mL ^− 1) with a 20 s response time. The method is inexpensive, highly sensitive, rapid and therefore provides a promising technology for on-site detection of BPA in food and water samples.

A. AC electrokinetics effect plays a vital role in BPA detection by introducing microfluidic movement to accelerate the molecular transport to the electrode surface.

B. The ACEK capacitive aptasensor has a limit of detection as low as 10 fM (2.8 fg ⋅ mL ^− 1) with a 20-s response time.

     

A self-assembled fusion protein-based surface plasmon resonance biosensor for rapid diagnosis of severe acute respiratory syndrome

A surface plasmon resonance (SPR)-based biosensor was developed for simple diagnosis of severe acute respiratory syndrome (SARS) using a protein created by genetically fusing gold binding polypeptides (GBPs) to a SARS coronaviral surface antigen (SCVme). The GBP domain of the fusion protein serves as an anchoring component onto the gold surface, exploiting the gold binding affinity of the domain, whereas the SCVme domain is a recognition element for anti-SCVme antibody, the target analyte in this study. SPR analysis indicated the fusion protein simply and strongly self-immobilized onto the gold surface, through GBP, without surface chemical modification, offering a stable and specific sensing platform for anti-SCVme detection. AFM and SPR imaging analyses demonstrated that anti-SCVme specifically bound to the fusion protein immobilized onto the gold-micropatterned chip, implying that appropriate orientation of bound fusion protein by GBP resulted in optimal exposure of the SCVme domain to the assay solution, resulting in efficient capture of anti-SCVme antibody. The best packing density of the fusion protein onto the SPR chip was achieved at the concentration of 10 μg mL⁻¹; this density showed the highest detection response (906 RU) for anti-SCVme. The fusion protein-coated SPR chip at the best packing density had a lower limit of detection of 200 ng mL⁻¹ anti-SCVme within 10 min and also allowed selective detection of anti-SCVme with significantly low responses for non-specific mouse IgG at all tested concentrations. The fusion protein provides a simple and effective method for construction of SPR sensing platforms permitting sensitive and selective detection of anti-SCVme antibody.     

Frontal analysis microchip capillary electrophoresis to study the binding of ligands to receptors derivatized on magnetic beads

The model binding of the glycopeptide antibiotic teicoplanin (Teic) from Actinoplanes teichomyceticus, immobilized on magnetic microspheres, to d-Ala-d-Ala terminus peptides was assessed using microchip capillary electrophoresis (MCE) with continuous frontal analysis (FA). Teic is closely related to vancomycin (Van), historically, the drug of last resort used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to the d-Ala-d-Ala terminus on the cell wall of Gram-positive bacteria via hydrogen bonds, thereby preventing the enzyme-mediated cross-linking of peptidoglycan and eventual cell death. In this work direct and competitive bead-based assays in a microfluidic chip are demonstrated. The binding constants obtained using the technique are comparable with values reported in the literature.     

A surface plasmon resonance sensor on a compact disk-type microfluidic device

A surface plasmon resonance (SPR) sensor on a compact disk (CD)-type microfluidic device was developed to miniaturize the elements of a complete analytical system, pump and valves. The CD-type microfluidic device was fabricated by attaching a polydimethylsiloxane disk plate that contained microchannels and reservoirs to a flat polycarbonate disk plate that contained grating films with a thin layer of Au. The optical system of the SPR sensor and the theory for its operation are based on the principle of a grating coupled-type SPR. The sample and reagent solutions in the reservoirs on the CD-type microfluidic device were sequentially introduced into the detection chamber by centrifugal force generated by the rotation of the microfluidic device. The variation of resonance wavelength was dependent on the refractive index of the sample solution. This CD-type SPR sensor was successfully used in an immunoassay of immunoglobulin A (IgA). The anti-IgA, blocking reagent, sample and washing solution in the reservoirs were sequentially introduced into the detection chamber by changing the frequency of rotation of the microfluidic device. IgA in the sample solution was adsorbed to the anti-IgA immobilized on the Au thin layer in the detection chamber and was then detected by the SPR sensor.     

Development of Novel Quantum Dots as Fluorescent Sensors for Application in Highly Sensitive Spectrofluorimetric Determination of Cu2+

Abstract

Quantum dots (QDs), semiconductor particles that have all three dimensions confined to the nanometer length scale are a good choice for the detection of heavy metals in aqueous media. In this study, novel CdSe/CdS QDs modified by mercaptoethanol were synthesized for Cu²⁺ detection, which shows high selectivity and sensitivity towards Cu²⁺ in the presence of other biological metallic ions. The detection mainly depends on the binding of Cu²⁺ onto the surface of QDs resulting in a chemical displacement of Cd²⁺. The subsequent formation of CuSe, and its application in hair and tea samples was also performed successfully.     

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (K _D ) in the range 2.2 × 10⁻¹¹ to 1.6 × 10⁻¹⁰ M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications.     

Microfluidic device for immunoassays based on surface plasmon resonance imaging

We have designed and fabricated a polydimethylsiloxane (PDMS) microfluidic device containing an array of gold spots onto which antigens or antibodies of interest can be attached. We use surface plasmon resonance (SPR) imaging to monitor the antibody-antigen recognition and binding events. This combination offers two significant advantages: (1) the microfluidic device dramatically reduces reaction time and sample consumption; and (2) the SPR imaging yields real-time detection of the immunocomplex formation. Thus, an immunoreaction may be detected and quantitatively characterized in about 10 min. The sensitivity of this method is at the subnanomolar level. When gold nanoparticles are selectively coupled to the immunocomplex to cause signal amplification, the sensitivity reaches the ten to one hundred picomolar level but the time required increases to about 60 min.     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.