Bacteriophage-Based Bioconjugates as a Flow Cytometry Probe for Fast Bacteria Detection

Robust detection of bacteria can significantly reduce risks of nosocomial infections, which are a serious problem even in developed countries (4.1 million cases each year in Europe). Here we demonstrate utilization of novel multifunctional bioconjugates as specific probes for bacteria detection. Bifunctional magnetic-fluorescent microparticles are coupled with bacteriophages. The T4 bacteriophage, due to its natural affinity to bacterial receptors, namely, OmpC and LPS, enables specific and efficient detection of Escherichia coli bacteria. Prepared probes are cheap, accessible (even in nonbiological laboratories), as well as versatile and easily tunable for different bacteria species. The magnetic properties of the bioconjugates facilitate the separation of captured target bacteria from other components of complex samples and other bacteria strains. Fluorescence enables simple analysis. We chose flow cytometry as the detection method as it is fast and widely used for biotests. The capture efficiency of the prepared bioconjugates is close to 100% in the range of bacteria concentrations from tens to around 10⁵ CFU/mL. The limit of detection is restricted by flow cytometry capabilities and in our case was around 10⁴ CFU/mL

Effect of hAM CCM on HUVECs migration assayed by scratch test.

<p>There are results of 160 measurements, 8 independent assays with 10 measurements for test and control each. Median values and (P25, P75) are shown (n = 8, p < 0.05). Detailed description of the assay is in Material and methods.</p

Growth factors in hAM CCM.

<p>Forty-one growth factors were quantitated by antibody array and the obtained values were combined into following growth factor families: EGF family (EGF-2, HB-EGF, EGF-R); FGF family (bFGF, FGF-4, FGF-6, FGF-7); Hematopoietic factors HF (MCSF, MCSF-R, SCF, SCF-R); IGF family (IGF-1, IGF-2, IGF-1SR); IGFBP family (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6); Neurotrophic factors NF (bNGF, GDNF, NT-3, NT-4); PDGF family (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-Ra, PDGF-Rb); TGF family (TGF-α, TGF-β, TGF-β2, TGF-β3); Vasculogenic factors VF (PLGF, VEGF, VEGF-R3, VEGF-D, VEGF-R2); some growth factors are presented separately: AR; G-CSF; GM-CSF; HGF. Each growth factor fluorescence value (FV) was measured and calculated as described in Materials and methods. (n = 4) (p < 0.05).</p

The example of real time migration assay of stimulated and controlled HUVECs directly from the X-Celligence system.

<p>The assay was repeated seven times with similar result during 25h observation at 37^°C. The difference between migration curves for cells in cultures with presence of hAM CCM and in control medium was significant. (p < 0.05).</p

Effect of hAM CCM on chemotaxy indeks of BM MNCs.

<p>The chemotaxy index (CI) after 2.5 h at 37^°C incubation time was calculated by dividing the number of cells in lower chamber by the number of cells added to the upper chamber counted at the start of the test. Median values and interquartile range (P25, P75) are shown (n = 12, p < 0.05).</p

Effect of hAM CCM on HUVECs expression of cell adhesion molecule-1 (CD31) marker.

<p>Significantly lower expression is observed in presence of hAM CCM. Median values and interquartile ranges (P25, P75) are shown (n = 3, p < 0.05).</p