Detection of Biomarkers of Periodontal Disease in Human Saliva Using Stabilized, Vertical Flow Immunoassays.

We report methods for stabilizing cellulose-based immunoassays and using this platform to analyze human saliva. Stabilization treatments of immunoassays for matrix metalloproteinases (MMP)-8 and -9, biomarkers of periodontal disease, were conducted and compared, revealing that anti-MMP-8 and -9 capture antibodies could be stabilized with the addition of a 5% trehalose solution to the test zones, followed by drying in a vacuum oven. After stabilization, the paper devices retained equivalent binding activity to that of freshly prepared tests for 14 days-a time frame that enables US-based clinical testing of this diagnostic assay. A saliva pretreatment method was developed to remove viscous elements without reducing the concentration or binding activity of dissolved proteins. Immunoassays were stored in ziplock bags containing desiccant, and used to detect nanomolar concentrations of MMP-9 in human saliva across the relevant clinical concentration range. These methods and findings facilitate rapid, affordable validation studies of this and other biomarkers that are found in saliva using vertical flow immunoassays.

Portable, Constriction-Expansion Blood Plasma Separation and Polymerization-Based Malaria Detection.

A portable, microfluidic blood plasma separation device is presented featuring a constriction-expansion design, which produces 100.0% purity for undiluted blood at 9% yield. This level of purity represents an improvement of at least 1 order of magnitude with increased yield compared to that achieved previously using passive separation. The system features high flow rates, 5-30 µL/min plasma collection, with minimal clogging and biofouling. The simple, portable blood plasma separation design is hand-driven and can easily be incorporated with microfluidic or laboratory scale diagnostic assays. The separation system was applied to a paper-based diagnostic test for malaria that produced an amplified color change in the presence of Plasmodium falciparum histidine-rich protein 2 at a concentration well below clinical relevancy for undiluted whole blood.

Assessment of colorimetric amplification methods in a paper-based immunoassay for diagnosis of malaria.

Colorimetric detection methods that produce results readable by eye are important for diagnostic tests in resource-limited settings. In this work, we have compared three main types of colorimetric methods - enzymatic reactions, silver deposition catalyzed by gold nanoparticles, and polymerization-based amplification - in a paper-based immunoassay for detection of Plasmodium falciparum histidine-rich protein 2, a biomarker of malarial infection. We kept the binding events in the immunoassay constant in order to isolate the effect of the detection method on the outcome of the test. We have highlighted that the optimal readout time in a test can vary significantly - ranging from immediately after addition of a visualization agent to 25 minutes after addition of a visualization agent - depending on the colorimetric method being used, and accurate time keeping is essential to prevent false positives in methods where substantial color develops over time in negative tests. We have also shown that the choice of a colorimetric method impacts the calculated limit-of-detection, the ease of visual perception of the readout, and the total cost of the assay, and therefore directly impacts the feasibility and the ease-of-use of a test in field settings.

A Method for Designing Instrument-Free Quantitative Immunoassays.

Colorimetric readouts are widely used in point-of-care diagnostic immunoassays to indicate either the presence or the absence of an analyte. For a variety of reasons, it is more difficult to quantify rather than simply detect an analyte using a colorimetric test. We report a method for designing, with minimal iteration, a quantitative immunoassay that can be interpreted objectively by a simple count of number of spots visible to the unaided eye. We combined a method called polymerization-based amplification (PBA) with a series of microscale features containing a decreasing surface density of capture molecules, and the central focus of the study is understanding how the choice of surface densities impacts performance. Using a model pair of antibodies, we have shown that our design approach does not depend on measurement of equilibrium and kinetic binding parameters and can provide a dynamic working range of 3 orders of magnitude (70 pM to 70 nM) for visual quantification.

Polymerization-based signal amplification for paper-based immunoassays.

Diagnostic tests in resource-limited settings require technologies that are affordable and easy to use with minimal infrastructure. Colorimetric detection methods that produce results that are readable by eye, without reliance on specialized and expensive equipment, have great utility in these settings. We report a colorimetric method that integrates a paper-based immunoassay with a rapid, visible-light-induced polymerization to provide high visual contrast between a positive and a negative result. Using Plasmodium falciparum histidine-rich protein 2 as an example, we demonstrate that this method allows visual detection of proteins in complex matrices such as human serum and provides quantitative information regarding analyte levels when combined with cellphone-based imaging. It also allows the user to decouple the capture of analyte from signal amplification and visualization steps.