Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays.

The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture the target nucleic acid sequence with an immobilized complementary strand anchored to a substrate, and then to visualize this event using a complementary label nucleic acid bound to a nanoparticle label. A critical component of high-sensitivity nucleic acid detection is to utilize high-density capture surfaces for the effective capture of target nucleic acid. Multiple methods have been reported, including the use of streptavidin-based protein anchors that can be adsorbed to the lateral flow substrate and that can utilize the high-affinity streptavidin-biotin linkage to bind biotinylated nucleic acid capture sequences for subsequent target nucleic acid binding. However, these protein anchors have not been systematically characterized for use in the context of nucleic acid detection. In this work, we characterize several protein-based anchors on nitrocellulose for (i) capturing the robustness of the attachment of the protein anchor, (ii) capturing nucleic acid density, and (iii) targeting nucleic acid capture. Further, we demonstrate the signal gains in target nucleic acid hybridization made by increasing the density of capture nucleic acid on a nitrocellulose substrate using multiple applications of protein loading onto nitrocellulose. Finally, we use our high-density capture surfaces to demonstrate high-sensitivity nucleic acid detection in a lateral flow assay (in the context of a SARS-CoV-2 sequence), achieving a LOD of approximately 0.2 nM.

Comparison of signal enhancement strategies for carbamazepine detection in undiluted human saliva using an electrochemical sensor with stencil-printed carbon electrodes.

Carbamazepine (CBZ), a drug prescribed to prevent seizures in people with epilepsy, has a narrow therapeutic range such that patients would greatly benefit from personalized drug dosage recommendations. Saliva is an excellent sample for personalized monitoring of CBZ levels because saliva CBZ concentration correlates with the free concentration of CBZ in blood, and can be collected non-invasively. CBZ level quantification using electrochemical detection has been demonstrated in a variety of electrode systems and samples, however, human saliva presents a particular challenge in terms of its complex composition that can result in signal interference via a high background current at the potentials of interest for CBZ detection. Previous demonstrations of electrochemical detection of CBZ in saliva have included rigorous pre-treatment of the sample using centrifugation and high levels of dilution, which is not compatible with lower-resource field settings for patient monitoring of CBZ levels. In this work, we systematically investigate several strategies to improve the detection of CBZ in a background of undiluted human saliva using polymeric laminate-based devices with stencil-printed carbon electrodes; (i) adding the anionic surfactant sodium dodecyl sulfate to the saliva, (ii) filtering saliva to remove larger molecular weight species, (iii) plasma pretreatment of the device electrodes, and (iv) incubation of the sample on the electrodes. These methods enabled the quantification of therapeutically-relevant concentrations of CBZ in a background of human saliva without the need for saliva preprocessing like dilution.

A survey of 3D printing technology applied to paper microfluidics.

Paper microfluidics is a rapidly growing subfield of microfluidics in which paper-like porous materials are used to create analytical devices that are well-suited for use in field applications. 3D printing technology has the potential to positively affect paper microfluidic device development by enabling tools and methods for the creation of devices with well-defined and tunable fluidic networks of porous matrices for high performance signal generation. This critical review focuses on the progress that has been made in using 3D printing technologies to advance the development of paper microfluidic devices. We describe printing work in three general categories: (i) solid support structures for paper microfluidic device components; (ii) channel barrier definition in existing porous materials; and (iii) porous channels for capillary flow, and discuss their value in advancing paper microfluidic device development. Finally, we discuss major areas of focus for highest impact on the next generation of paper microfluidics devices.

Dry storage of multiple reagent types within a paper microfluidic device for phenylalanine monitoring.

The degradation of biochemical reagents on the timescale of weeks can severely limit the utility of microfluidic assays intended for field use, and is a challenging aspect of microfluidic device development in general. Our study focuses on the evaluation of the dry storage stability of three types of reagents: (i) the colorimetric reagents nitroblue tetrazolium and 1-methoxy-5-methylphenazinium methylsulfate, (ii) the enzyme phenylalanine dehydrogenase, and (iii) the coenzyme ß-nicotinamide adenine dinucleotide hydrate, within the context of a phenylalanine monitoring device. We have demonstrated stable dry storage of each of the reagents, over the time span of approximately one month. Drying the colorimetric reagents under nitrogen was found to largely suppress reagent degradation and the appearance of nonspecific signal, while the enzyme and coenzyme retained activity when stored dry for a month without additional processing or chemical additives. Finally, phenylalanine monitoring devices with all three reagent types dried down and stored for 15 days showed comparable functionality to devices containing freshly-dried reagents - a key milestone to enable future clinical testing.

Rational design and characterization of a lateral flow assay for canine C-reactive protein in wound exudate.

C-reactive protein levels may have clinical value in monitoring different phases of healing and in identifying possible states of infection. As a critical step to further investigate this potential connection, we have demonstrated a lateral flow assay (LFA) for canine CRP level assessment in wound exudate that could be used as a tool in the veterinary clinic setting. In the rational design of our cCRP LFA, we have characterized LFA performance for sequential delivery mode vs. the more common premixed delivery mode using several metrics including dynamic range, sensitivity, limit of detection, and time to result. Although the sequential mode assay results indicated modestly improved signal (3-14%) and limit of detection (in the low ng/mL range for both) for this set of cCRP immunoassay reagents, the premixed mode assay's shorter run time with one less delivery step was chosen for use in this application in which analyte levels are substantially elevated. We have defined a straightforward wound exudate processing procedure that includes centrifugation to extract exudate from canine patient bandages, and subsequent sample dilution for cCRP quantification by our LFA. And, we have demonstrated that our cCRP LFA provides comparable cCRP concentrations to that of gold-standard ELISA performed on the same clinical wound exudate, and serum/plasma samples. Finally, we have highlighted some next steps in the assessment of cCRP as a biomarker for wound healing and infection.

Characterization methods in porous materials for the rational design of multi-step processing in the context of a paper microfluidic phenylalanine test.

A promising application of paper microfluidics is the translation of gold-standard multi-step laboratory tests to a disposable paper-based format for decentralized diagnostic or therapeutic testing. This often entails conversion of bench-top processing of macro-volume samples to the processing of micro-volume samples within a porous matrix, and requires detailed characterization of fluid and reagent interactions within the porous material(s) of the device. The current study focuses on rational device design through the characterization of fluid and reagent interactions in polysulfone and glass fiber substrates for multi-step sample processing. Specifically, we demonstrate how the characterization of fluidic compatibility between substrates, chemical compatibility between reagents and substrates, sample pH, and sample transport can be used to inform device design in the context of a two-reaction detection scheme for phenylalanine in porous materials. Finally, we demonstrate detection of phenylalanine from human whole blood, and discuss the multiple strengths of the current design over a previous version.

Integrated wax valve for robust fluid control in an electrochemical fabric-based device.

Although fabrics have great promise as substrates for use in wearable precision health applications, there has been relatively little attention focused on the development of control tools suitable for use in fabrics. Fluid control tools in fabric would enable the automation of multi-step sample processing on the device, reducing the need for off-chip sample handling. In this study, we describe the operation and characterization of a wax-based valving method with an integrated resistive heater in fabric for automated fluid delivery. The combination of wax-transfer-printed wax barrier and stencil-printed conductive ink heating element in fabric is a novel approach for achieving fluid control. We demonstrate robust valve operation and a rapid valve response time, and quantify the reproducibility of fluid flow through replicate valves. Further, we characterize wax redistribution in fabric using optical methods and scanning electron microscope imaging. Finally, we demonstrate valve utility in the context of on-device incubation in a fabric-based device for electrochemical glucose sensing. With a fabrication method that is compatible with a variety of substrates, this valving method has broad applicability.

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 107 and 1.34 × 107 CEID50/mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Disposable Autonomous Device for Swab-to-Result Diagnosis of Influenza.

A prototype of a self-contained, automated, disposable device for chemically amplified protein-based detection of influenza virus from nasal swab specimens was developed and evaluated in a clinical setting. The device required only simple specimen manipulation without any dedicated instrumentation or specialized training by the operator for interpretation. The device was based on a sandwich immunoassay for influenza virus nucleoprotein; it used an enzyme-labeled antibody and a chromogenic substrate to provide an amplified visible signal, in a two-dimensional paper network format. All reagents were stored within the device. Device performance was assessed at Seattle Children's Hospital; clinical staff collected nasal swab samples from 25 patients and then operated test devices on site to detect influenza A and B in those specimens. The total test time from device initiation to result was approximately 35 min. Device performance for influenza A detection was ∼70% accurate using in-house qRT-PCR influenza A as a gold-standard comparison. The ratio of valid to total completed device runs yielded a success rate of 92%, and the negative predictive value for both the influenza A and B assay was 81%. The ability to diagnose respiratory infections rapidly and close to the patient was well received by hospital staff, inspiring further optimization of device function.

Progress in the development and integration of fluid flow control tools in paper microfluidics.

Paper microfluidics is a rapidly growing subfield of microfluidics in which paper-like porous materials are used to create analytical devices. There is a need for higher performance field-use tests for many application domains including human disease diagnosis, environmental monitoring, and veterinary medicine. A key factor in creating high performance paper-based devices is the ability to manipulate fluid flow within the devices. This critical review is focused on the progress that has been made in (i) the development of fluid flow control tools and (ii) the integration of those tools into paper microfluidic devices. Further, we strive to be comprehensive in our presentation and provide historical context through discussion and performance comparisons, when possible, of both relevant earlier work and recent work. Finally, we discuss the major areas of focus for fluid flow methods development to advance the potential of paper microfluidics for high-performance field applications.

Investigation of Reagent Delivery Formats in a Multivalent Malaria Sandwich Immunoassay and Implications for Assay Performance.

Conventional lateral flow tests (LFTs), the current standard bioassay format used in low-resource point-of-care (POC) settings, have limitations that have held back their application in the testing of low concentration analytes requiring high sensitivity and low limits of detection. LFTs use a premix format for a rapid one-step delivery of premixed sample and labeled antibody to the detection region. We have compared the signal characteristics of two types of reagent delivery formats in a model system of a sandwich immunoassay for malarial protein detection. The premix format produced a uniform binding profile within the detection region. In contrast, decoupling the delivery of sample and labeled antibody to the detection region in a sequential format produced a nonuniform binding profile in which the majority of the signal was localized to the upstream edge of the detection region. The assay response was characterized in both the sequential and premix formats. The sequential format had a 4- to 10-fold lower limit of detection than the premix format, depending on assay conjugate concentration. A mathematical model of the assay quantitatively reproduced the experimental binding profiles for a set of rate constants that were consistent with surface plasmon resonance measurements and absorbance measurements of the experimental multivalent malaria system.

Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Development of a Whole Blood Paper-Based Device for Phenylalanine Detection in the Context of PKU Therapy Monitoring.

Laboratory-based testing does not allow for the sufficiently rapid return of data to enable optimal therapeutic monitoring of patients with metabolic diseases such as phenylketonuria (PKU). The typical turn-around time of several days for current laboratory-based testing is too slow to be practically useful for effective monitoring or optimizing therapy. This report describes the development of a rapid, paper-based, point-of-care device for phenylalanine detection using a small volume (40 µL) of whole blood. The quantitative resolution and reproducibility of this device with instrumented readout are described, together with the potential use of this device for point-of-care monitoring by PKU patients.

A versatile valving toolkit for automating fluidic operations in paper microfluidic devices.

Failure to utilize valving and automation techniques has restricted the complexity of fluidic operations that can be performed in paper microfluidic devices. We developed a toolkit of paper microfluidic valves and methods for automatic valve actuation using movable paper strips and fluid-triggered expanding elements. To the best of our knowledge, this is the first functional demonstration of this valving strategy in paper microfluidics. After introduction of fluids on devices, valves can actuate automatically after a) a certain period of time, or b) the passage of a certain volume of fluid. Timing of valve actuation can be tuned with greater than 8.5% accuracy by changing lengths of timing wicks, and we present timed on-valves, off-valves, and diversion (channel-switching) valves. The actuators require ~30 µl fluid to actuate and the time required to switch from one state to another ranges from ~5 s for short to ~50 s for longer wicks. For volume-metered actuation, the size of a metering pad can be adjusted to tune actuation volume, and we present two methods - both methods can achieve greater than 9% accuracy. Finally, we demonstrate the use of these valves in a device that conducts a multi-step assay for the detection of the malaria protein PfHRP2. Although slightly more complex than devices that do not have moving parts, this valving and automation toolkit considerably expands the capabilities of paper microfluidic devices. Components of this toolkit can be used to conduct arbitrarily complex, multi-step fluidic operations on paper-based devices, as demonstrated in the malaria assay device.

Conversion of a laboratory-based test for phenylalanine detection to a simple paper-based format and implications for PKU screening in low-resource settings.

Laboratory-based testing does not reach many individuals in lower-resource settings who could benefit from access to appropriate tests for diagnosis and therapy. A critical issue is laboratory-based testing often requires an environment with a high level of resources and supporting infrastructure that is not available in many areas of the world. The current report describes the conversion of a laboratory-based test for phenylalanine detection to a simple paper-based test appropriate for use in low-resource settings. The paper-based test is easy to operate, with all reagents stored dry on the card, is compatible with visible detection for clinically relevant concentrations of phenylalanine, and has a time to result of 10 minutes. Next steps for test development are discussed in the context of the potential for the paper-based Phe test to be used as a newborn PKU screening test in settings that are not well served by existing screening approaches.

Enabling robust quantitative readout in an equipment-free model of device development.

A critical constraint in the design of appropriate medical devices for the lowest-resource settings is the lack of access to maintenance or repair on instrumentation. There are numerous point-of-care applications for which quantitative readout would have clinical utility. Thus, a challenge to the device developer is to enable quantitative device readout in an equipment-free model that is appropriate for use in even the lowest-resource settings. Paper microfluidics has great potential for enabling equipment-free devices that are low-cost, operable by minimally-trained users, and provide quantitative readout. The focus of this critical review is to describe the work, starting several decades ago and continuing to the present, to enable assays with quantitative readout in a fully-disposable device.

Long-term dry storage of an enzyme-based reagent system for ELISA in point-of-care devices.

Lateral flow devices are commonly used for many point-of-care (POC) applications in low-resource settings. However, they lack the sensitivity needed for many analytes relevant in the diagnosis of diseases. One approach to achieve higher sensitivity is signal amplification, which is commonly used in laboratory assays, but uses reagents that require refrigeration and inherently requires multiple assay steps not normally compatible with POC settings. Enzyme-based signal amplification, such as the one used in ELISA, could greatly improve the limit of detection if it were translated to a format compatible with POC requirements. A signal-amplified POC device not only requires the reagents to be stored in a stable form, but also requires automation of the multiple sequential steps of signal amplification protocols. Here, we describe a method for the long-term dry storage of ELISA reagents: horseradish peroxidase (HRP) conjugated antibody label and its colorimetric substrate diaminobenzidine (DAB). The HRP conjugate retained ∼80% enzymatic activity after dry storage at 45 °C for over 5 months. The DAB substrate was also stable at 45 °C and exhibited no detectable loss of activity over 3 months. These reagents were incorporated into a two-dimensional paper network (2DPN) device that automated the steps of ELISA for the detection of a malarial biomarker. These results demonstrate the potential of enzyme-based signal amplification for enhanced sensitivity in POC devices for low resource settings.

Tunable-delay shunts for paper microfluidic devices.

We demonstrate a novel method for controlling fluid flow in paper-based devices. The method delays fluid progress through a porous channel by diverting fluid into an absorbent pad-based shunt placed into contact with the channel. Parameters to control the delay include the length and the thickness of the shunt. Using this method, reproducible delays ranging from 3 to 20 min were achieved. A simple electrical circuit model was presented and used to predict the delays in a system. Results from the model showed good agreement with experimental observations. Finally, the shunts were used for the sequential delivery of fluids to a detection zone in a point-of-care compatible folding card device using biochemical reagents for the amplified detection of the malaria protein PfHRP2.

Progress in the development of paper-based diagnostics for low-resource point-of-care settings.

This Review focuses on recent work in the field of paper microfluidics that specifically addresses the goal of translating the multistep processes that are characteristic of gold-standard laboratory tests to low-resource point-of-care settings. A major challenge is to implement multistep processes with the robust fluid control required to achieve the necessary sensitivity and specificity of a given application in a user-friendly package that minimizes equipment. We review key work in the areas of fluidic controls for automation in paper-based devices, readout methods that minimize dedicated equipment, and power and heating methods that are compatible with low-resource point-of-care settings. We also highlight a focused set of recent applications and discuss future challenges.

Dissolvable bridges for manipulating fluid volumes in paper networks.

A capability that is key to increasing the performance of paper microfluidic devices is control of fluid transport in the devices. We present dissolvable bridges as a novel method of manipulating fluid volumes within paper-based devices. We demonstrate and characterize the operation of the bridges, including tunability of the volumes passed from 10 µL to 80 µL, using parameters such as geometry and composition. We further demonstrate the utility of dissolvable bridges in the important context of automated delivery of different volumes of a fluid from a common source to multiple locations in a device for simple device loading and activation.