Bacteriophage Imaging Immunoassay for Point of Care Diagnostics
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Point-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.