Gregory G. Lewis

Quantifying Analytes in Paper‐Based Microfluidic Devices Without Using External Electronic Readers

Point-of-care (POC) and point-of-use assays are critical for identifying and measuring the quantity of analytes in a variety of environments that lack access to laboratory infrastructure. In quantitative versions of these assays, both the duration of the assay and the output signal must be measured. Measurements of time most often are performed using a timer that is external to the platform of the assay. Such measurements are relatively simple and inexpensive, and in some cases, can be integrated into the device itself. In contrast, measurements of signal typically are accomplished using hand-held electrochemical, absorbance, reflectance, transmittance, or fluorescence readers, and as such, these measurements can be complicated, time-consuming, and expensive, particularly in the context of extremely resource-limited environments such as remote villages in the developing world. The World Health Organization has identified the use of external readers as a challenge that must be overcome when creating ideal POC diagnostic assays for use in the developing world. In fact, they have listed “equipment-free” as one of seven necessary attributes for diagnostic tests in these regions. Herein, we describe two complimentary assay strategies that address this issue. By using paper-based microfluidic devices, we show that the level of an analyte can be quantified by simply measuring time: no external electronic reader is required for the quantitative measurement (Figure 1). The methods involve either 1) tracking the time required for a sample to react with and ultimately pass through a hydrophobic detection reagent in a single conduit within a threedimensional (3D) paper-based microfluidic device (Figure 1a) (we call this a digital assay), or 2) counting the number of bars that become colored after a fixed assay period in a related paper-based microfluidic device (Figure 1b; we refer to this as an analog assay). The methods described herein require only a timer, the ability to see color, and/or the

Quantitative Fluorescence Assays Using a Self-Powered Paper-Based Microfluidic Device and a Camera-Equipped Cellular Phone.

Fluorescence assays often require specialized equipment and, therefore, are not easily implemented in resource-limited environments. Herein we describe a point-of-care assay strategy in which fluorescence in the visible region is used as a readout, while a camera-equipped cellular phone is used to capture the fluorescent response and quantify the assay. The fluorescence assay is made possible using a paper-based microfluidic device that contains an internal fluidic battery, a surface-mount LED, a 2-mm section of a clear straw as a cuvette, and an appropriately-designed small molecule reagent that transforms from weakly fluorescent to highly fluorescent when exposed to a specific enzyme biomarker. The resulting visible fluorescence is digitized by photographing the assay region using a camera-equipped cellular phone. The digital images are then quantified using image processing software to provide sensitive as well as quantitative results. In a model 30 min assay, the enzyme β-D-galactosidase was measured quantitatively down to 700 pM levels. This Communication describes the design of these types of assays in paper-based microfluidic devices and characterizes the key parameters that affect the sensitivity and reproducibility of the technique.

Point-of-Care Assay Platform for Quantifying Active Enzymes to Femtomolar Levels Using Measurements of Time as the Readout

This Article describes a strategy for quantifying active enzyme analytes in a paper-based device by measuring the time for a reference region in the paper to turn green relative to an assay region. The assay requires a single step by the user, yet accounts for variations in sample volume, assay temperature, humidity, and contaminants in a sample that would otherwise prevent a quantitative measurement. The assay is capable of measuring enzymes in the low to mid femtomolar range with measurement times that range from ~30 s to ~15 min (lower measurement times correspond to lower quantities of the analyte). Different targets can be selected in the assay by changing a small molecule reagent within the paper-based device, and the sensitivity and dynamic range of the assays can be tuned easily by changing the composition and quantity of a signal amplification reagent or by modifying the configuration of the paper-based microfluidic device. By tuning these parameters, limits-of-detection for assays can be adjusted over an analyte concentration range of low femtomolar to low nanomolar, with dynamic ranges for the assays of at least 1 order of magnitude. Furthermore, the assay strategy is compatible with complex fluids such as serum.

Two general designs for fluidic batteries in paper-based microfluidic devices that provide predictable and tunable sources of power for on-chip assays

Microfluidic devices fabricated out of paper (and paper and tape) have emerged as promising platforms for conducting multiple diagnostic assays simultaneously in resource-limited settings. Certain types of assays in these devices, however, require a source of power to function. Lithium ion, nickel-cadmium, and other types of batteries have been used to power these devices, but these traditional batteries are too expensive and pose too much of a disposal hazard for diagnostic applications in resource-limited settings. To circumvent this problem, we previously designed a “fluidic battery” that is composed of multiple galvanic cells, incorporated directly into a multilayer paper-based microfluidic device. We now show that multiple cells of these fluidic batteries can be connected in series and/or in parallel in a predictable way to obtain desired values of current and potential, and that the batteries can be optimized to last for a short period of time (<1 min) or for up to 10–15 min. This paper also (i) outlines and quantifies the parameters that can be adjusted to maximize the current and potential of fluidic batteries, (ii) describes two general configurations for fluidic batteries, and (iii) provides equations that enable prediction of the current and potential that can be obtained when these two general designs are varied. This work provides the foundation upon which future applications of fluidic batteries will be based.

The expanding role of paper in point-of-care diagnostics

This editorial discusses the expanding role of paper as a platform on which to build new point-of-care assays, particularly those intended for use in resource-limited settings. Successful diagnostics for use in these environments require a low-cost platform (possibly paper) as well as new assay strategies, reagents and materials for achieving selectivity and sensitivity. Paper provides a common platform for bringing these components together and serves as a low-cost medium for prototyping new point-of-care assays.

Depolymerizable poly(benzyl ether)-based materials for selective room temperature recycling

This communication addresses the question of whether it is possible to design a polymer that can be modified easily to create various traditional classes of polymeric materials while also incorporating a mechanism into the backbone of the polymer for facilitating selective end-of-life recycling capabilities into the new types of materials. We illustrate these capabilities using depolymerizable poly(benzyl ethers), which we modify to access desired properties in plastics. The poly(benzyl ethers) also are designed for selective, programmed, room temperature, and continuous depolymerization of plastics to monomers when the plastic is no longer needed.

Self‐Immolative Poly(4,5‐dichlorophthalaldehyde) and its Applications in Multi‐Stimuli‐Responsive Macroscopic Plastics

End-capped poly(4,5-dichlorophthalaldehyde) (PCl2PA), which is a new self-immolative CD(r) polymer with the unique capability of depolymerizing continuously and completely in the solid state when an end cap is cleaved from the polymer by reaction with a specific molecular signal, is described. End-capped poly(4,5-dichlorophthalaldehyde) is sufficiently stable to enable patterning of three-dimensional macroscopic polymeric materials by selective laser sintering. These unique materials are capable of 1) autonomously amplifying macroscopic changes in the material in response to specific molecular inputs, and 2) altering their responses depending on the identity of the applied signal. Thus, not only does end-capped PCl2PA provide new and unique capabilities compared to the small subset of existing CD(r) polymers, but it also provides access to a new class of stimuli-responsive materials.

A strategy for minimizing background signal in autoinductive signal amplification reactions for point-of-need assays

Rapid point-of-need assays are used to detect abundant biomarkers. The development of in situ signal amplification reactions could extend these assays to screening and triaging of patients for trace levels of biomarkers, even in resource-limited settings. We, and others, have developed small molecule-based in situ signal amplification reactions that eventually may be useful in this context. Herein we describe a design strategy for minimizing background signal that may occur in the absence of the target analyte, thus moving this in situ signal amplification approach one step closer to practical applications. Specifically, we describe allylic ethers as privileged connectors for linking detection and propagating functionality in a small molecule signal amplification reagent. Allylic ethers minimize background reactions while still enabling controlled release of a propagating signal in order to continue the signal amplification reaction. This paper characterizes the ability of allylic ethers to provide an amplified response, and offers insight into additional design considerations that are needed before in situ small molecule-based signal amplification becomes a viable strategy for point-of-need diagnostics.

Quantitative Point-of-Care (POC) Assays Using Measurements of Time as the Readout: A New Type of Readout for mHealth

A paper-based microfluidic device was used to quantitatively detect active enzyme analytes in samples at mid to low femtomolar levels. The device uses a hydrophobic oligomer that controls the wetting properties of the paper within the device. When the target analyte is present within the sample, the oligomer depolymerizes, thus switching the paper to hydrophilic, allowing for the sample to wick through the device. Measuring the time for the sample to wick to a control region relative to an assay region within the device results in sensitive, quantitative measurements of the target enzyme (e.g., alkaline phosphatase or β-D-galactosidase). This device requires the use of only a timer for quantifying a target analyte, and thus the platform may be appropriate for use in resource-limited environments, where access to expensive diagnostic equipment is limited. A smartphone with integrated application software (the software has yet to be developed) could be used for timing the assay and for relating the time measurement to the quantitative readout for the assay. In future versions of this assay, it should be possible to configure the smartphone to start and stop the time-based measurement to further simplify the assay for the user.