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A method for designing instrument-free, quantitative immunoassays Shefali Lathwal, and Hadley D. Sikes Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04538 • Publication Date (Web): 15 Feb 2016 Downloaded from http://pubs.acs.org on February 18, 2016
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Analytical Chemistry
A METHOD FOR DESIGNING INSTRUMENT-FREE, QUANTITATIVE IMMUNOASSAYS Shefali Lathwal and Hadley D. Sikes* Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ABSTRACT: 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 three orders of magnitude (70 pM to 70 nM) for visual quantification.
INTRODUCTION Assays that rely on visual, colorimetric readouts are useful in point-of-care (POC) diagnostic testing, especially in resource-limited settings (RLS) where laboratory infrastructure is not accessible. Such tests are widely used to provide robust qualitative results.1,2 Quantitative information about analyte levels can be valuable3 in assessing the severity of disease and for monitoring the effectiveness of treatment, but it is more difficult to obtain in colorimetric tests. To quantify analyte levels, tests typically require visual comparison with reference color charts2 or additional analysis of colorimetric intensity through imaging and image processing using scanners,4 cameras,5 cellphones6 or customized readers7. However, the requirement of additional processing steps and the potential for changes in ambient light during imaging to affect the quantitative analysis6 are disadvantages of imaging-based approaches in RLS. The use of reference color charts, though instrument-free, introduces subjectivity during visual comparison of colors.8,9 Therefore, a need exists for simple quantitative diagnostic tests in RLS that can be interpreted objectively using the unaided eye. Equipment-free, visual quantification methods connect analytes in a variety of ways with chemical reactions that produce a colored product and have recently been reviewed.10 Quantification is accomplished by visually interpreting the difference in intensity or color of readout (intensity or hue-based readout),8,11 measuring the length of a colored interfacial region produced (distance based readout),12–15 measuring the time required for the appearance of color,16,17 or measuring the time interval between
two different colored appearances18 (time-based readout), and measuring the number of colored bars16,17 or colored lines19–23 that appear after a specified time (ladder-bar readout). The distance-based and ladder-bar approaches have been used in immunoassays, where antibodies are used to capture and facilitate detection of analytes through specific, molecular recognition events. Since immunoassays are one of the most common types of diagnostic tests used in RLS,3,24 this class of assays is particularly important. Immunoassays typically use detection antibodies conjugated to either metal colloids (i.e. gold)3 or enzymes4 to generate a colorimetric readout. Previous reports of visual, quantitative immunoassays involved a lateral flow format where a sample containing an analyte passed sequentially over several test lines containing an identical concentration of a capture antibody or an area uniformly coated with capture antibody, and either the number of colored lines visible to the unaided eye19–22 or the length of the colored region12 produced after a specified time indicated the quantity of analyte. Existing visual quantitative immunoassays have reported a dynamic working range (i.e., the range between the lowest and the highest concentration of analyte that can be distinctly quantified) of one order of magnitude (one log).12,19– 21 In practice, the clinically relevant dynamic range of analytes that are detected using immunoassays can vary over several logs.25–27 For example, C-Reactive Protein (CRP) levels can vary from 200 μg mL-1 associated with severe bacterial infections and burns.30 The wide range has resulted in two separate semiquantitative commercial tests for measuring levels between 1-10 μg mL-1 and 10-500 μg mL-1. Therefore, we posed the question whether design, combined with an appropriate colorimetric detection technology, can be used to improve upon the dynamic range of visual quantification in a single test. In addition to providing a broad dynamic range, the detection technology that is chosen to generate a colorimetric readout should also be capable of measuring small concentrations of the analyte (
