Increasing Binding Efficiency via Reporter Shape and Flux in a Viral Nanoparticle Lateral-Flow Assay

To identify factors controlling the performance of reporter particles in a sensitive lateral-flow assay (LFA), we investigated the effect of the flux and shape of filamentous bacteriophage (phage) on the performance of phage LFAs. Phage of three different lengths and diameters were modified with biotin and AlexaFluor 555 as binding and read-out elements, respectively. The binding efficiencies of the functionalized phage were tested in a fibrous glass LFA membrane modified with avidin. The total binding rate, quantified using real-time particle counting and particle image velocimetry, decreased monotonically with the average bulk flux of phage through the membrane. At the pore scale, more phage bound in regions with faster local flow, confirming that both average and local flux increased binding. The number of bound phage increased with the aspect ratio of the phage and scaled with the phage surface area, consistent with a binding interaction controlled by the number of recognition elements on the surface. Together, these results indicate that increasing the likelihood that recognition elements on the surface of phage encounter the fibers enhances the assay binding efficiency and suggests one origin for the improved performance of nonspherical phage reporters

Development of a panel of recombinase polymerase amplification assays for detection of common bacterial urinary tract infection pathogens

Aims: To develop and evaluate the performance of a panel of isothermal real?time recombinase polymerase amplification (RPA) assays for detection of common bacterial urinary tract infection (UTI) pathogens. Methods and Results: The panel included RPAs for Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Enterococcus faecalis. All five RPAs required reaction times of under 12 min to reach their lower limit of detection of 100 genomes per reaction or less, and did not cross?react with high concentrations of nontarget bacterial genomic DNA. In a 50?sample retrospective clinical study, the five?RPA assay panel was found to have a specificity of 100% (95% CI, 78�0%) and a sensitivity of 89% (95% CI, 75�%) for UTI detection. Conclusions:The analytical and clinical validity of RPA for the rapid and sensitive detection of common UTI pathogens was established. Significance and Impact of the Study: Rapid identification of the causative pathogens of UTIs can be valuable in preventing serious complications by helping avoid the empirical treatment necessitated by traditional urine culture's 48�?h turnaround time. The routine and widespread use of RPA to supplement or replace culture?based methods could profoundly impact UTI management and the emergence of multidrug?resistant pathogens

Orientational binding modes of reporters in a viral-nanoparticle lateral flow assay

Using microscopy and image analysis, we characterize binding of filamentous viral nanoparticles to a fibrous affinity matrix as models for reporter capture in a lateral flow assay (LFA). M13 bacteriophage (M13) displaying an in vivo-biotinylated peptide (AviTag) genetically fused to the M13 tail protein p3 are functionalized with fluorescent labels. We functionalize glass fiber LFA membranes with antibodies to M13, which primarily capture M13 on the major p8 coat proteins, or with avidin, which captures M13 at the biotin-functionalized tail, and compare orientational modes of reporter capture for the side- versus tip-binding recognition interactions. The number of captured M13 is greater for side-binding than for tip-binding, as expected from the number of recognition groups. Whereas two-thirds of side-bound M13 captured by an anti-M13 antibody bind immediately after colliding with the membrane, tip-bound M13 prominently exhibit three additional orientational modes that require M13 to reorient to enable binding. These results are consistent with the idea that the elongated M13 shape couples with the complex flow field in an open and disordered fibrous LFA membrane to enhance capture

Helium beam shadowing for high spatial resolution patterning of antibodies on microstructured diagnostic surfaces

We have developed a technique for the high-resolution, self-aligning, and high-throughput patterning of antibody binding functionality on surfaces by selectively changing the reactivity of protein-coated surfaces in specific regions of a workpiece with a beam of energetic helium particles. The exposed areas are passivated with bovine serum albumin (BSA) and no longer bind the antigen. We demonstrate that patterns can be formed (1) by using a stencil mask with etched openings that forms a patterned exposure, or (2) by using angled exposure to cast shadows of existing raised microstructures on the surface to form self-aligned patterns. We demonstrate the efficacy of this process through the patterning of anti-lysozyme, anti-Norwalk virus, and anti-Escherichia coli antibodies and the subsequent detection of each of their targets by the enzyme-mediated formation of colored or silver deposits, and also by binding of gold nanoparticles. The process allows for the patterning of three-dimensional structures by inclining the sample relative to the beam so that the shadowed regions remain unaltered. We demonstrate that the resolution of the patterning process is of the order of hundreds of nanometers, and that the approach is well-suited for high throughput patterning

Detection and Monitoring of Microparticles Under Skin by Optical Coherence Tomography as an Approach to Continuous Glucose Sensing Using Implanted Retroreflectors

We demonstrate the feasibility of using optical coherence tomography (OCT) to image and detect 2.8 ?m diameter microparticles (stationary and moving) on a highly-reflective gold surface both in clear media and under skin in vitro. The OCT intensity signal can clearly report the microparticle count, and the OCT response to the number of microparticles shows a good linearity. The detect ability of the intensity change (2.9%�5%) caused by an individual microparticle shows the high sensitivity of monitoring multiple particles using OCT. An optical sensing method based on this feasibility study is described for continuously measuring blood sugar levels in the subcutaneous tissue, and a molecular recognition unit is designed using competitive binding to modulate the number of bound microparticles as a function of glucose concentration. With further development, an ultra-small, implantable sensor might provide high specificity and sensitivity for long-term continuous monitoring of blood glucose concentration

Association energetics of cross-reactive and specific antibodies

HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG1 antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally

Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation

We report a novel, modular approach to immuno-detection based on antibody recognition and PCR read-out that employs antibody-conjugated bacteriophage, easily-manipulated nonpathogenic viruses, as affinity agents. Our platform employs phage genetically tagged for in vivo biotinylation during phage maturation that can easily be linked, through avidin, to any biotinylatable affinity agent, including full-length antibodies, peptides, lectins or aptamers. The presence of analyte is reported with high sensitivity through real-time PCR. This approach avoids the need to clone antibody-encoding DNA fragments, allows the use of full-length, high affinity antibodies and, by having DNA reporters naturally encapsulated inside the bacteriophage, greatly reduces nonspecific binding of DNA. We validate the efficacy of this new approach through the detection of VEGF (Vascular Endothelial Growth Factor), a known angiogenic cancer biomarker protein, at attomolar concentrations in bronchoalveolar lavage (BAL) fluid

Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays

We introduce the modification of bacteriophage particles with aptamers for use as bioanalytical reporters, and demonstrate the use of these particles in ultrasensitive lateral flow assays. M13 phage displaying an in vivo biotinylatable peptide (AviTag) genetically fused to the phage tail protein pIII were used as reporter particle scaffolds, with biotinylated aptamers attached via avidin朾iotin linkages, and horseradish peroxidase (HRP) reporter enzymes covalently attached to the pVIII coat protein. These modified viral nanoparticles were used in immunochromatographic sandwich assays for the direct detection of IgE and of the penicillin-binding protein from Staphylococcus aureus (PBP2a). We also developed an additional lateral flow assay for IgE, in which the analyte is sandwiched between immobilized anti-IgE antibodies and aptamer-bearing reporter phage modified with HRP. The limit of detection of this LFA was 0.13 ng/mL IgE, ?100 times lower than those of previously reported IgE assays