Tissue biopsy and swab culture are the gold standards for diagnosing tissue infection; these tests require significant time, diagnostic costs, and resources. Towards earlier and specific diagnosis of infection, a non-destructive, rapid, and mobile detection device is described to distinguish bacterial species via light scatter spectra from the surface of an infected tissue, reagent-free. Porcine skin and human cadaveric skin models of wound infection were used with a 650 nm LED and an angular photodiode array to detect bacterial infections on the tissue surface, which can easily be translated to a typical CMOS array or smartphone. Tissue samples were inoculated with Escherichia coli, Salmonella Typhimurium, or Staphylococcus aureus and backscatter was collected from 100° to 170° in 10° increments; each bacterial species resulted in unique Mie scatter spectra. Distinct Mie scatter spectra were obtained from epidermis (intact skin model) and dermis (wound model) samples, as well as from porcine and human cadaveric skin samples. Interactions between bacterial colonies and lipid particles within dermis samples generated a characteristic Mie scatter spectrum, while the lipid itself did not contribute to such characteristic spectrum as corroborated with body lotion experiments. The designed angular photodiode array is able to immediately and non-destructively detect tissue bacterial infection and identify the species of infection within three seconds, which could greatly improve point of care diagnostics and antibiotic treatments.
Cryptosporidium parvum is a difficult-to-detect protozoan that causes diarrhea in the healthy adults and death in
immunocompromised individuals. While it is easy to understand the transmission routes of Cryptosporidium, it is
currently difficult to identify low concentrations of Cryptosporidium, especially when following EPA method 1623,
which can easily require tens of liters of water to get a positive signal. The current detection method is unacceptable and
severely inefficient when taking into account the time that goes into concentrating a sample, actual assays, and training
associated with the assays. Using our method, it is possible to use only 15 μL of sample, which is an
immunoagglutination assay that uses Mie scatter intensity changes to detect different Cryptosporidium concentrations. In
addition to creating a standard curve using a clean sample matrix (i.e., phosphate buffered saline), field samples were
collected from a chlorine treated swimming pool, a sump located on a farm, and a turtle pond. Each sample had different
intensity changes but the trend represented within the data was the same. This assay has a detection limit of 100-101
oocysts/mL and can be done in as little as 10 minutes.
Virus antigens of avian influenza subtype H3N2 were detected on two different microfluidic platforms: microchannel
and droplet. Latex immunoagglutination assays were performed using 920-nm highly carboxylated polystyrene beads
that are conjugated with antibody to avian influenza virus. The bead suspension was merged with the solutions of avian
influenza virus antigens in a Y-junction of a microchannel made by polydimethylsiloxane soft lithography. The resulting
latex immunoagglutinations were measured with two optical fibers in proximity setup to detect 45° forward light
scattering. Alternatively, 10 μL droplets of a bead suspension and an antigen solution were merged on a
superhydrophobic surface (water contact angle = 155°), whose movement was guided by a metal wire, and 180° back
light scattering is measured with a backscattering optical probe. Detection limits were 0.1 pg mL-1 for both microchannel
with proximity fibers and droplet microfluidics, thanks to the use of micro-positioning stages to help generate
reproducible optical signals. Additionally, optical waveguide was tested by constructing optical waveguide channels
(filled with mineral oil) within a microfluidic device to detect the same light scattering. Detection limit was 0.1 ng mL-1
for an optical waveguide device, with a strong potential of improvement in the near future. The use of optical waveguide
enabled smaller device setup, easier operation, smaller standard deviations and broader linear range of assay than
proximity fiber microchannel and droplet microfluidics. Total assay time was less than 10 min.
An alternative approach for fabricating a protein array at nanoscale (<100 nm) is suggested with a capability of
characterization and/or localization of multiple components on a nanoarray. Basically, fluorescent micro- and
nanospheres each conjugated with different proteins are size-dependently self-assembled (SDSA) onto these nanometer
wells that were created on the polymethyl methacrylate (PMMA) substrate by electron beam lithography (EBL).
Particles of different diameters are added serially, and electrostatically attached to the corresponding wells through
electrostatic attraction between the carboxylic groups of the spheres and p-doped silicon substrate underneath the
PMMA layer. This SDSA was enhanced by wire-guide manipulation of droplets on the surface containing nanometer
wells. Target detection utilizes fluorescence resonance energy transfer (FRET) from fluorescent beads to target (mouse
immunoglobulin G = mIgG or Octamer-4 = Oct4) and its antibody bound on the beads. The 180 nm blue beads are
conjugated with mIgG to capture anti-mIgG-FITC. The 50 nm green and 100 nm yellow-green beads are conjugated
with anti-Oct4 to capture Oct4 peptides; where the secondary anti-Oct4 tagged with phycoerythrin via F(ab)2 fragment
is then added to function as an indicator of Oct4 detection. These protein-conjugated particles are added serially from the
largest to the smallest and the particles are successfully self-assembled to the respective nanometer wells to achieve sizedependent
self-assembly. FRET signals are detected through fluorescence and confocal microscopes, and further
confirmed by Fluorolog3 spectrofluorometer. Therefore, SDSA is a valuable approach for the fabrication of multiple
components array; and FRET is a useful biorecognition technique for the detection of mIgG, Oct4 or other targets of
interest.
We have investigated the utilization of particle agglutination assays using forward light scattering measurements in a
microfluidic device towards detecting viral particles. The model viral target was bovine viral diarrhea virus (BVDV).
Highly carboxylated polystyrene microspheres (510 nm) were coated with anti-BVDV monoclonal antibodies. This
solution was in turn used to detect live modified BVDV. This assay was first performed in a two well slide for proof of
concept and then in a simple y-channel microfluidic device with optical fibers arranged in a close proximity setup.
Particle immunoagglutination was detected through static light scattering measurements taken at 45° to incident light. In
the microfluidic device, modified live BVDV was detected with a detection limit of 0.5 TCID50 mL-1.
Microfluidic device detections of E. coli K12 in deionized (DI) water and E. coli in field water sample were
demonstrated through static light scattering of latex immunoagglutination using proximity optical fibers. This method is
a fully-automated, one-step detection, and requires neither sample pre-treatment nor cell culturing often required in
many on-chip detections. We have used highly carboxylated polystyrene submicron latex particles without surfactants to
enhance diffusional mixing and prevent non-specific bindings towards successful demonstration of latex
immunoagglutination in microfluidic device. Detection of E. coli was performed by taking microscopic images from the
view cell of a microfluidic device and counting the fractions of non-agglutinated and agglutinated particles. The limit of
detection (LOD) was ca. 150 CFU ml-1 with this method for both E. coli K12 in DI water and E. coli in field water
sample, indicating no non-specific bindings. Improved LOD of < 4.3 CFU ml-1 was achieved by measuring forward
static light scattering from microfluidic device, using proximity optical fibers and a USB-powered miniature
spectrometer. The total assay time for sample preparation (mostly dilutions) and on-chip assay (mostly injections and
short incubation time) was < 10 min.
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