Two-Fluorophore Mobile Phone Imaging of Biplexed Real-Time NAATs Overcomes Optical Artifacts in Highly Scattering Porous Media.

Nucleic acid amplification tests (NAATs) are common in laboratory and clinical settings because of their low time to result and exquisite sensitivity and specificity. Laboratory NAATs include onboard positive controls to reduce false negatives and specialized hardware to enable real-time fluorescence detection. Recent efforts to translate NAATs into at-home tests sacrifice one or more of the benefits of laboratory NAATs, such as sensitivity, internal amplification controls (IACs), or time to result. In this manuscript, we describe a mobile-phone-based strategy for real-time imaging of biplexed NAATs in paper. The strategy consisted of: (1) using mobile phones with multipass excitation and emission filters on the flash and camera to image the signal from distinct fluorophore-labeled probe types in a biplexed NAAT in a glass fiber membrane; and (2) analyzing the differential fluorescence signal between the red and green color channels of phone images to overcome a strong evaporation-induced optical artifact in heated glass fiber pads due to changes in the refractive index. We demonstrated that differential fluorescence imaging enabled low limits of detection (316 copies of methicillin-resistant Staphylococcus aureus DNA) in our lab's "MD NAAT" platform, even in biplexed isothermal strand displacement amplification reactions containing 100k copies of coamplifying IAC DNA templates. These results suggest that two-fluorophore mobile phone imaging may enable translating the benefits of extant laboratory-based, real-time NAATs to the point of care.

Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices.

Nucleic acid amplification test (NAAT)-based point-of-care (POC) devices are rapidly growing for use in low-resource settings. However, key challenges are the ability to store the enzyme-based reagents in dry form in the device and the long-term stability of those reagents at elevated temperatures, especially where ambient temperatures could be as high as 45 °C. Here, we describe a set of excipients including a combination of trehalose, polyethylene glycol and dextran, and a method for using them that allows long-term dry storage of enzyme-based reagents for an isothermal strand displacement amplification (iSDA) reaction in a porous matrix. Various porous materials, including nitrocellulose, cellulose, and glass fiber, were tested. Co-dried reagents for iSDA always included those that amplified the ldh1 gene in Staphylococcus aureus (a polymerase and a nicking enzyme, 4 primers, dNTPs and a buffer). Reagents also either included a capture probe and a streptavidin-Au label required for lateral flow (LF) detection after amplification, or a fluorescent probe used for real-time detection. The reagents showed the best stability in a glass fiber matrix when stored in the presence of 10% trehalose and 2.5% dextran. The reagents were stable for over a year at ∼22 °C as determined by lateral flow detection and gel electrophoresis. The reagents also exhibited excellent stability after 360 h at 45 °C; the assay still detected as few as 10 copies of ldh1 gene target by lateral flow detection, and 50 copies with real-time fluorescence detection. These results demonstrate the potential for incorporation of amplification reagents in dry form in point-of-care devices for use in a wide range of settings.

Mobile Phone Ratiometric Imaging Enables Highly Sensitive Fluorescence Lateral Flow Immunoassays without External Optical Filters.

Paper-based diagnostic tests based on the lateral flow immunoassay concept promise low-cost, point-of-care detection of infectious diseases, but such assays suffer from poor limits of detection. One factor that contributes to poor analytical performance is a reliance on low-contrast chromophoric optical labels such as gold nanoparticles. Previous attempts to improve the sensitivity of paper-based diagnostics include replacing chromophoric labels with enzymes, fluorophores, or phosphors at the expense of increased fluidic complexity or the need for device readers with costly optoelectronics. Several groups, including our own, have proposed mobile phones as suitable point-of-care readers due to their low cost, ease of use, and ubiquity. However, extant mobile phone fluorescence readers require costly optical filters and were typically validated with only one camera sensor module, which is inappropriate for potential point-of-care use. In response, we propose to couple low-cost ultraviolet light-emitting diodes with long Stokes-shift quantum dots to enable ratiometric mobile phone fluorescence measurements without optical filters. Ratiometric imaging with unmodified smartphone cameras improves the contrast and attenuates the impact of excitation intensity variability by 15×. Practical application was shown with a lateral flow immunoassay for influenza A with nucleoproteins spiked into simulated nasal matrix. Limits of detection of 1.5 and 2.6 fmol were attained on two mobile phones, which are comparable to a gel imager (1.9 fmol), 10× better than imaging gold nanoparticles on a scanner (18 fmol), and >2 orders of magnitude better than gold nanoparticle-labeled assays imaged with mobile phones. Use of the proposed filter-free mobile phone imaging scheme is a first step toward enabling a new generation of highly sensitive, point-of-care fluorescence assays.

Wavelengths and Lifetimes of Paper Autofluorescence: A Simple Substrate Screening Process to Enhance the Sensitivity of Fluorescence-Based Assays in Paper.

Porous media made of nitrocellulose and glass fiber are common "paper" substrates for lateral flow assays, microfluidic paper analytical devices and other point-of-care diagnostic assays. Such assays commonly use optical labels such as gold nanoparticles, latex beads, or fluorescent nanoparticles to visualize the presence of analytes. Fluorescent labels are commonly used in bioassays to enhance sensitivity, but autoluminescence of the paper substrate worsens signal-to-noise ratios of fluorescence-based assays. To date, there exists no systematic investigation of autoluminescence wavelengths or lifetimes of porous membranes used in lateral flow assays. In response, we quantified the autoluminescence of commonly used porous materials across the visible spectrum via excitation-emission spectroscopy and time-resolved fluorescence spectroscopy, and demonstrate that autoluminescence is solely due to autofluorescence with lifetimes of about 5 ns in the visible spectrum. Counterintuitively, we found that spectroscopy alone does not provide sufficient information to select candidate paper substrates for fluorophore-labeled assays. Therefore, we developed a simple quantitative framework to select a low-fluorescence substrate that minimizes both the overlap of paper and fluorophore emission spectra and the fluorescence intensity on an imaging system of interest (such as a gel imager). Use of this framework was shown to lower the limit of detection of an influenza A nucleoprotein immunoassay by over 50%. The tools developed in this manuscript enable assay developers to screen appropriate, low-fluorescence porous substrates and enhance the sensitivity of membrane-based fluorescence assays.

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.

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.

Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis.

We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.

Highly sensitive immunoassay based on controlled rehydration of patterned reagents in a 2-dimensional paper network.

We have demonstrated a multistep 2-dimensional paper network immunoassay based on controlled rehydration of patterned, dried reagents. Previous work has shown that signal enhancement improves the limit of detection in 2-dimensional paper network assays, but until now, reagents have only been included as wet or dried in separate conjugate pads placed at the upstream end of the assay device. Wet reagents are not ideal for point-of-care because they must be refrigerated and typically limit automation and require more user steps. Conjugate pads allow drying but do not offer any control of the reagent distribution upon rehydration and can be a source of error when pads do not contact the assay membrane uniformly. Furthermore, each reagent is dried on a separate pad, increasing the fabrication complexity when implementing multistep assays that require several different reagents. Conversely, our novel method allows for consistent, controlled rehydration from patterned reagent storage depots directly within the paper membrane. In this assay demonstration, four separate reagents were patterned in different regions of the assay device: a gold-antibody conjugate used for antigen detection and three different signal enhancement components that must not be mixed until immediately before use. To show the viability of patterning and drying reagents directly onto a paper device for dry reagent storage and subsequent controlled release, we tested this device with the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) as an example of target analyte. In this demonstration, the signal enhancement step increases the visible signal by roughly 3-fold and decreases the analytical limit of detection by 2.75-fold.

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.

UbiNAAT: a multiplexed point-of-care nucleic acid diagnostic platform for rapid at-home pathogen detection.

The COVID-19 pandemic increased demands for respiratory disease testing to facilitate treatment and limit transmission, demonstrating in the process that most existing test options were too complex and expensive to perform in point-of-care or home scenarios. Lab-based molecular techniques can detect viral RNA in respiratory illnesses but are expensive and require trained personnel, while affordable antigen-based home tests lack sensitivity for early detection in newly infected or asymptomatic individuals. The few home RNA detection tests deployed were prohibitively expensive. Here, we demonstrate a point-of-care, paper-based rapid analysis device that simultaneously detects multiple viral RNAs; it is demonstrated on two common respiratory viruses (COVID-19 and influenza A) spiked onto a commercial nasal swab. The automated device requires no sample preparation by the user after insertion of the swab, minimizing user operation steps. We incorporated lyophilized amplification reagents immobilized in a porous matrix, a novel thermally actuated valve for multiplexed fluidic control, a printed circuit board that performs on-device lysis and amplification within a cell-phone-sized disposable device. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) products are visualized via fluorescent dyes using a modified cell phone, resulting in detection of as few as 104 viral copies per swab across both pathogens within 30 minutes. This integrated platform could be commercialized in a form that would be inexpensive, portable, and sensitive; it can readily be multiplexed to detect as many as 8 different RNA or DNA sequences, and adapted to any desired RNA or DNA detection assays.

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.

Disposable platform for bacterial lysis and nucleic acid amplification based on a single USB-powered printed circuit board.

Recent advances in electronics and microfluidics have enabled several research groups to develop fully integrated, sample-to-result isothermal nucleic acid amplification test (NAAT) platforms for the point of care. However, high component counts and costs have limited translation of these platforms beyond the clinic to low-resource settings-including homes. Many NAATs include complex, multi-component heater electronics based on flex circuits or multiple printed circuit boards (PCBs) to support essential NAAT steps such as lysis, sample deactivation, and nucleic acid amplification. In contrast, current commercial assays for home use, such as those for pregnancy or ovulation that include electronics, typically have just one onboard PCB. This work describes a generalizable strategy to integrate all heaters and the electronics needed to control them onto a single low-cost, USB-powered PCB. We built a multiplexable disposable NAAT ("MD NAAT") platform that applies these principles, integrating small-area heaters that heat small regions to near-boiling (for pathogen lysis and deactivation) and large-area heaters (for amplification) on the same PCB. We show that both classes of heaters have high intra-board and inter-device reproducibility despite only heating a NAAT cartridge from below. We validated the small-area heaters by lysing methicillin-resistant Staphylococcus aureus (MRSA) cells and the large-area heaters by performing two types of isothermal NAATs (isothermal strand displacement amplification (iSDA) and loop-mediated isothermal amplification (LAMP)). These results demonstrate the merit of integrating NAAT heaters and control electronics onto a single printed circuit board and are a step toward translating NAATs to the home.

Two-dimensional paper network format that enables simple multistep assays for use in low-resource settings in the context of malaria antigen detection.

The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

Microfluidic diagnostic technologies for global public health.

The developing world does not have access to many of the best medical diagnostic technologies; they were designed for air-conditioned laboratories, refrigerated storage of chemicals, a constant supply of calibrators and reagents, stable electrical power, highly trained personnel and rapid transportation of samples. Microfluidic systems allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developed-world laboratory. These systems must be inexpensive, but also accurate, reliable, rugged and well suited to the medical and social contexts of the developing world.

Near-digital amplification in paper improves sensitivity and speed in biplexed reactions.

The simplest point-of-care assays are usually paper and plastic devices that detect proteins or nucleic acids at low cost and minimal user steps, albeit with poor limits of detection. Digital assays improve limits of detection and analyte quantification by splitting a sample across many wells (or droplets), preventing diffusion, and performing analyte amplification and detection in multiple small wells. However, truly digital nucleic acid amplification tests (NAATs) require costly consumable cartridges that are precisely manufactured, aligned, and operated to enable low detection limits. In this study, we demonstrate how to implement near-digital NAATs in low-cost porous media while approaching the low limits of detection of digital assays. The near-digital NAAT was enabled by a paper membrane containing lyophilized amplification reagents that automatically, passively meters and distributes a sample over a wide area. Performing a NAAT in the paper membrane while allowing diffusion captures many of the benefits of digital NAATs if the pad is imaged at a high spatial resolution during amplification. We show that the near-digital NAAT is compatible with a low-cost paper and plastic disposable cartridge coupled to a 2-layer rigid printed circuit board heater (the MD NAAT platform). We also demonstrate compatibility with biplexing and imaging with mobile phones with different camera sensors. We show that the near-digital NAAT increased signal-to-noise ratios by ~ 10×, improved limits of detection from above 103 copies of methicillin-resistant Staphylococcus aureus genomic DNA to between 100 and 316 copies in a biplexed reaction containing 105 copies of co-amplifying internal amplification control DNA, and reduced time-to-result from 45 min of amplification to 15-20 min for the positive samples.

Morel-Lavallée Lesions: A Diagnostic and Clinical Dilemma.

Morel-Lavallée lesions (MLL) occur following closed degloving injuries in trauma and appear as subcutaneous fluid collections. These lesions provide a diagnostic dilemma due to their resemblance to other subcutaneous lesions, including post-traumatic or post-procedural hematomas, and malignant entities such as soft tissue sarcomas. Magnetic resonance imaging (MRI) remains the diagnostic test of choice to diagnose MLL. Here we present a single case of MLL in a 40-year-old female who sustained an injury to her left thigh months earlier after falling through a pier as well as a review of literature on the diagnostic findings typically seen in this type of injury.

Enhanced sensitivity of lateral flow tests using a two-dimensional paper network format.

Point-of-care diagnostic assays that are rapid, easy-to-use, and low-cost are needed for use in low-resource settings; the lateral flow test has become the standard bioassay format in such settings because it meets those criteria. However, for a number of analytes, conventional lateral flow tests lack the sensitivity needed to have clinical utility. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions. The two-dimensional structures allow incorporation of multistep processes for improved sensitivity, while still retaining the positive aspects of conventional lateral flow tests. Here we create an easy-to-use, signal-amplified immunoassay based on a modified commercial strip test for human chorionic gonadotropin, the hormone used to detect pregnancy, and demonstrate an improved limit of detection compared to a conventional lateral flow assay. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

"Smart" diblock copolymers as templates for magnetic-core gold-shell nanoparticle synthesis.

We report a new strategy for synthesizing temperature-responsive gamma-Fe(2)O(3)-core/Au-shell nanoparticles (Au-mNPs) from diblock copolymer micelles. The amphiphilic diblock copolymer chains were synthesized using reversible addition-fragmentation chain-transfer (RAFT) with a thermally responsive "smart" poly(N-isopropylacrylamide) (pNIPAAm) block and an amine-containing poly(N,N-dimethylaminoethylacrylamide) (DMAEAm) block that acted as a reducing agent during gold shell formation. The Au-mNPs reversibly aggregated upon heating the solution above the transition temperature of pNIPAAm, resulting in a red-shifted localized surface plasmon resonance.

Visualization and measurement of flow in two-dimensional paper networks.

The two-dimensional paper network (2DPN) is a versatile new microfluidic format for performing complex chemical processes. For chemical detection, for example, 2DPNs have the potential to exceed the capabilities and performance of existing paper-based lateral flow devices at a comparable cost and ease of use. To design such 2DPNs, it is necessary to predict 2D flow patterns and velocities within them, but because of the scattering of the paper matrix, conventional particle imaging velocimetry is not practical. In this note, we demonstrate two methods for visualization of flow in 2DPNs that are inexpensive, easy to implement, and quantifiable.