Fast and Highly Sensitive Detection of Pathogens Wreathed with Magnetic Nanoparticles Using Dark-Field Microscopy

Cryptosporidium
parvum (C. parvum)
is a highly potent zoonotic pathogen,
which can do significant harm to both human beings and livestock.
However, existing technologies or methods are deficient for rapid
on-site detection of water contaminated with C. parvum. Better detection approaches are needed to allow water management
agencies to stop major breakouts of the pathogen. Herein, we present
a novel detection method for cryptosporidium in a tiny drop of sample
using a magnetic nanoparticle (MNP) probe combined with dark-field
microscopy in 30 min. The designed MNP probes bind with high affinity
to C. parvum, resulting in the formation
of a golden garland-like structure under dark-field microscopy. This
MNP-based dark-field counting strategy yields an amazing PCR-like
sensitivity of 8 attomolar (aM) (5 pathogens in 1 μL). Importantly,
the assay is very rapid (∼30 min) and is very simple to perform
as it involves only one step of mixing and magnetic separation, followed
by dropping on a slide for counting under dark-field microscope. By
combining the advantages of the specific light-scattering characteristic
of MNP probe under dark field and the selective magnetic separation
ability of functionalized MNP, the proposed MNP-based dark-field enumeration
method offers low cost and significant translational potential.