Browsing by Author "Kim, Jinsu"
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Item Aptamer-Phage Reporters for Ultrasensitive Lateral Flow Assays(Analytical Chemistry, 2016-12) Adhikari, Meena; Strych, Ulrich; Kim, Jinsu; Goux, Heather J.; Dhamane, Sagar; Poongavanam, Mohan-Vivekanandan; Hagstrom, Anna E. V.; Kourentzi, Katerina D.; Conrad, Jacinta C.; Willson, Richard C.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.Item Bacteriophage Imaging Immunoassay for Point of Care Diagnostics(2016-08) Kim, Jinsu; Conrad, Jacinta C.; Willson, Richard C.; Vekilov, Peter G.; Varadarajan, Navin; Shih, Wei-Chuan; Ghasemi, HadiPoint-of-care (PoC) devices are used for medical testing at or near the site of patient care. Due to its low cost, simple assay operation, and ease of mass production, the lateral flow immunoassay (LFA) is one of the most widely used and commercially available PoC tests. Nevertheless, traditional LFAs remain limited by two main issues: lack of sensitivity and difficulties in quantification. To develop sensitive and quantitative LFAs, we can consider three strategies (1) new LFA reaction membranes, (2) new reporter materials, and/or (3) new read-out methods. Here, we developed functionalized phage nanoparticles as a new sensitive reporter for LFAs. The use of phage as a scaffold for attachment of multiple bio-recognition and read-out-signal molecules constitutes a novel and innovative approach in LFAs. We first developed fluorescently labeled M13 phage that also are functionalized with anti-analyte antibodies. Individual phage bound to the target analyte (here MS2 virus as a model) and captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses. Next, to understand the high sensitivity, we characterized the binding modes of the phage reporter to targets in the fibrous glass LFA membrane using microscopy and image analysis. We found that the elongated shape of M13 phage coupled with the complex flow promotes reorientation and facilitates the binding. The binding efficiency was also influenced by other assay parameters, such as the length of the phage and their flux through the LFA membrane. The number of bound phage increased as the phage length increased; similarly the number of bound phage increased with the flux [within a particular flow regime]. These results suggested that the increased length and flux of phage increased the chance that phage encountered fibers, thereby enhancing binding efficiency. Next, as a first step towards practical phage LFAs we characterized the stability and durability of phage at elevated temperatures. To reveal the mechanism of temperature-tolerant mutant stability, we characterized the mutant genomes using next-generation sequencing technology. Three potential mechanisms were suggested for the apparent increase in temperature tolerance: gene replication enhancement (due to mutations in gp2); formation of miniphage; and mutations in the p7 coat protein. Finally, as a first step towards a user-friendly and handheld system compatible with PoC use, we incorporated two photon detectors, a multi-pixel photon counter (MPPC) and a photomultiplier tube (PMT), into a smartphone accessory. The sensitivities of those detectors were compared by determining a low level of 1,5-anhydroglucitol (AHG) as a model test reaction in a chemiluminescence assay. The assay sensitivity depended on the detector performance; the PMT detector exhibited ten-fold better sensitivity than the MPPC. These results raise the promising possibility that the developed detectors could be applied to our phage LFA by inserting the appropriate light source and optical filters.Item Detection of Viruses By Counting Single Fluorescent Genetically Biotinylated Reporter Immunophage Using a Lateral Flow Assay(ACS Appl Mater Interfaces, 2016-02) Kim, Jinsu; Adhikari, Meena; Dhamane, Sagar; Hagstrom, Anna E. V.; Kourentzi, Katerina D.; Strych, Ulrich; Willson, Richard C.; Conrad, Jacinta C.We demonstrated a lateral flow immunoassay (LFA) for detection of viruses using fluorescently labeled M13 bacteriophage as reporters and single-reporter counting as the readout. AviTag-biotinylated M13 phage were functionalized with antibodies using avidin朾iotin conjugation and fluorescently labeled with AlexaFluor 555. Individual phage bound to target viruses (here MS2 as a model) captured on an LFA membrane strip were imaged using epi-fluorescence microscopy. Using automated image processing, we counted the number of bound phage in micrographs as a function of target concentration. The resultant assay was more sensitive than enzyme-linked immunosorbent assays and traditional colloidal-gold nanoparticle LFAs for direct detection of viruses.Item Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays(Scientific Reports, 2016-04) Chen, Hui; Hagstrom, Anna E. V.; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew S.; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina D.; Conrad, Jacinta C.; Atmar, Robert L.; Willson, Richard C.In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 106 virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings.Item Increasing Binding Efficiency via Reporter Shape and Flux in a Viral Nanoparticle Lateral-Flow Assay(ACS Applied Materials and Interfaces, 2018-03) Kim, Jinsu; Vu, Binh V.; Kourentzi, Katerina D.; Willson, Richard C.; Conrad, Jacinta C.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.Item Orientational binding modes of reporters in a viral-nanoparticle lateral flow assay(Analyst, 2017-12) Kim, Jinsu; Poling-Skutvik, Ryan; Trabuco, Joao R. C.; Kourentzi, Katerina D.; Willson, Richard C.; Conrad, Jacinta C.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.