17 research outputs found
A paperfluidic platform to detect Neisseria gonorrhoeae in clinical samples
Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.This work was funded by the National Institute of Health National Institute of Allergy and Infectious Diseases award number R01 AI113927 to Boston University and the NIH National Institute of Biomedical and Bioengineering award number U54 EB007958 to Johns Hopkins University. (R01 AI113927 - National Institute of Health National Institute of Allergy and Infectious Diseases; U54 EB007958 - NIH National Institute of Biomedical and Bioengineering)Accepted manuscrip
Development and clinical validation of Iso-IMRS: a novel diagnostic assay for P. falciparum malaria
In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/μL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.Accepted manuscrip
Groepsgedrag op de nanoschaal
Monodisperse gas microbubbles, encapsulated with a shell
of photopolymerizable
diacetylene lipids and phospholipids, were produced by microfluidic
flow focusing, for use as ultrasound contrast agents. The stability
of the polymerized shell microbubbles against both aggregation and
gas dissolution under physiological conditions was studied. Polyethylene
glycol (PEG) 5000, which was attached to the diacetylene lipids, was
predicted by molecular theory to provide more steric hindrance against
aggregation than PEG 2000, and this was confirmed experimentally.
The polymerized shell microbubbles were found to have higher shell-resistance
than nonpolymerizable shell microbubbles and commercially available
microbubbles (Vevo MicroMarker). The acoustic stability under 7.5
MHz ultrasound insonation was significantly greater than that for
the two comparison microbubbles. The acoustic stability was tunable
by varying the amount of diacetylene lipid. Thus, our polymerized
shell microbubbles are a promising platform for ultrasound contrast
agents
A Mass-Tagging Approach for Enhanced Sensitivity of Dynamic Cytokine Detection Using a Label-Free Biosensor
Monitoring
cytokine release by cells allows the investigation of
cellular response to specific external stimuli, such as pathogens
or candidate drugs. Unlike conventional colorimetric techniques, label-free
detection of cytokines enables studying cellular secretions in real
time by eliminating additional wash and labeling steps after the binding
step. However, label-free techniques that are based on measuring mass
accumulation on a sensor surface are challenging for measuring small
cytokines binding to much larger capture agents (usually antibodies)
because the relative signal change is small. This problem is exacerbated
when the capturing antibodies desorb from the surface, a phenomenon
that almost inevitably occurs in immunoassays but is rarely accounted
for. Here, we demonstrate a quantitative dynamic detection of interleukine-6
(IL-6), a pro-inflammatory cytokine, using an interferometric reflectance
imaging sensor (IRIS). We improved the accuracy of the quantitative
analysis of this relatively small protein (21 kDa) by characterizing
the antibody desorption rate and compensating for the antibody loss
during the binding experiment. By correcting for protein desorption,
we achieved an analytical limit of detection at 19 ng/mL IL-6 concentration.
We enhanced the sensitivity by 7-fold by using detection antibodies
that recognize a different epitope of the cytokine. We demonstrate
that these detection antibodies, which we call “mass tags”,
can be used concurrently with the target analyte to eliminate an additional
wash and binding step. Finally, we report successful label-free detection
of IL-6 in cell culture medium (with 10% serum) with comparable signal
to that obtained in PBS. This work is the first to report quantitative
dynamic label-free detection of small protein in a complex biological
fluid using IRIS