Paper-based Immunoassays Research Articles

Cannabis detection with solid sensors and paper-based immunoassays by conjugating antibodies to nanocellulose

Highly sensitive and specific diagnostics for cannabis usage are essential for rapid on-site screening for illicit drug usage. To improve the sensitivity of THC immunoassays, a proper immobilization of the sensing elements on the sensor substrate is critical. In this work, we demonstrated the utilization of EDC/NHS coupling chemistry with nanocellulose to obtain efficient anchor layers for the immobilization of anti-immune complex antibodies on surfaces. In our approach, the high surface-to-volume ratio, OH-group-rich surface, and high hygroscopicity of TOCNF enable efficient surface functionalization and enhance water permeation inside the nanocellulose network structure, offering a hydrophilic spacer for the sensing antibodies. THC detection was shown in both SPR (surface plasmon resonance technique) and paper-based sensing systems. In SPR, antibody immobilization and the related interactions with the target molecule complex with 1–10 μg/mL THC were followed in-situ in aqueous environment, revealing robust attachment of the antibody to the nanocellulose layer and preserved bioactivity. Additionally, quantitative THC detection was enabled on paper substrate by colorimetric means by employing labeled anti-THC Fab antibody fragments as detection antibodies. THC detection efficiency of covalently linked biointerface was superior compared to the performance of physically linked biointerface. The chemical conjugation of anti-IC to nanocellulose allowed efficient binding, whereas supramolecular conjugation led to insufficient binding, highlighting the relevance of the developed nanocellulose-based anchor layer.

A review of cardiac troponin I detection by surface enhanced Raman spectroscopy: Under the spotlight of point-of-care testing

Cardiac troponin I (cTnI) is a biomarker widely related to acute myocardial infarction (AMI), one of the leading causes of death around the world. Point-of-care testing (POCT) of cTnI not only demands a short turnaround time for its detection but the highest accuracy levels to set expeditious and adequate clinical decisions. The analytical technique Surface-enhanced Raman spectroscopy (SERS) possesses several properties that tailor to the POCT format, such as its flexibility to couple with rapid assay platforms like microfluidics and paper-based immunoassays. Here, we analyze the strategies used for the detection of cTnI by SERS considering POCT requirements. From the detection ranges reported in the reviewed literature, we suggest the diseases other than AMI that could be diagnosed with this technique. For this, a section with information about cardiac and non-cardiac diseases with cTnI release, including their release kinetics or cut-off values are presented. Likewise, POCT features, the use of SERS as a POCT technique, and the biochemistry of cTnI are discussed. The information provided in this review allowed the identification of strengths and lacks of the available SERS-based point-of-care tests for cTnI and the disclosing of requirements for future assays design.

Towards quantitative point of care detection using SERS lateral flow immunoassays

The rapid detection of biomolecules in a point of care (POC) setting is very important for diagnostic purposes. A platform which can provide this, whilst still being low cost and simple to use, is paper-based lateral flow immunoassays (LFIA). LFIA combine immunology and chromatography to detect a target by forming an immunocomplex with a label which traps them in a test zone. Qualitative analysis can be performed using the naked eye whilst quantitative analysis takes place by measuring the optical signal provided by the label at the test zone. There are numerous detection methods available; however, many suffer from low sensitivity and lack of multiplexing capabilities or are poor at providing POC quantitative analysis. An attractive method to overcome this is to use nanoparticles coated in Raman reporters as the labelled species and to analyse test zones using surface-enhanced Raman scattering (SERS). Due to the wide variety of metal nanoparticles, Raman reporter and laser excitations that are available, SERS-based LFIA have been adapted to identify and quantify multiple targets at once. Large Raman microscopes combined with long mapping times have limited the platform to the lab; however, by transferring the analysis to portable Raman instruments, rapid and quantitative measurements can be taken at the POC without any loss in sensitivity. Portable or handheld SERS-LFIA platforms can therefore be used anywhere, from modern clinics to remote and resource-poor settings. This review will present an overview of SERS-based LFIA platforms and the major recent advancements in multiplexing and portable and handheld detection with an outlook on the future of the platform.Graphical abstract

Developing a Paper-Based Antigen Assay to Differentiate between Coronaviruses and SARS-CoV-2 Spike Variants.

COVID-19 first appeared in December of 2019 in Wuhan, China. Since then, it has become a global pandemic. A robust and scalable diagnostics strategy is crucial for containing and monitoring the pandemic. RT-PCR is a known, reliable method for COVID-19 diagnostics, which can differentiate between SARS-CoV-2 and other viruses. However, PCR is location-dependent, time-consuming, and relatively expensive. Thus, there is a need for a more flexible method, which may be produced in an off-the-shelf format and distributed more widely. Paper-based immunoassays can fulfill this function. Here, we present the first steps toward a paper-based test, which can differentiate between different spike proteins of various coronaviruses, SARS-CoV-1, SARS-CoV-2, and CoV-HKU1, with negligible cross-reactivity for HCoV-OC43 and HCoV-229E in a single assay, which takes less than 30 min. Furthermore, our test can distinguish between fractions of the same spike protein. This is done by an altered assay design with four test line locations where each antigen builds a unique, identifiable binding pattern. The effect of several factors, such as running media, immunoprobe concentration, and antigen interference, is considered. We find that running media has a significant effect on the final binding pattern where human saliva provides results while human serum leads to the lowest signal quality.

Carbohydrate binding module-fused antibodies improve the performance of cellulose-based lateral flow immunoassays

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.

The Immunoprobe Aggregation State is Central to Dipstick Immunoassay Performance.

As new infectious disease outbreaks become more likely, it is important to be able to develop and deploy appropriate testing in time. Paper-based immunoassays are rapid, cheap, and easy to produce at scale and relatively user friendly but often suffer from low selectivity and sensitivity. Understanding the molecular mechanisms of paper immunoassays may help improve and hasten development and therefore production and market availability. Here, we study how the behavior of nanoparticle-antibody immunoprobes in paper dipstick immunoassays is impacted by synthesis strategy and surface chemistry architecture. We conjugate gold nanoparticles to polyclonal anti-immunoglobulin G (IgG) and anti-zika NS1 antibodies by electrostatic adsorption and N-hydroxysuccinimide (NHS) and hydrazide (Hz) chemistries. The immunoprobes were used in paper immunoassays and the effective affinity for the antigen was quantified from the test line intensities, as well as the distribution of the immunoprobes throughout the strips. The results show that nanoparticle colloidal stability, both post synthesis and during antigen binding, is a key factor and affects immunoassay results and performance, often through reduction or loss of signal.

Functional comparison of paper-based immunoassays based on antibodies and engineered binding proteins.

Binding protein scaffolds, such as rcSso7d, have been investigated for use in diagnostic tests; however, the functional performance of rcSso7d has not yet been studied in comparison to antibodies. Here, we assessed the analyte-binding capabilities of rcSso7d and antibodies on cellulose with samples in buffer and 100% human serum.

Paper-Based Immunoassays Gain Currency

Genetic Engineering & Biotechnology NewsVol. 39, No. 8 OMICSPaper-Based Immunoassays Gain CurrencyConvenient and increasingly versatile, newly minted paper-based assays may win wide acceptance in both resource-constrained and resource-endowed settingsRichard A. Stein, MD, PhDRichard A. Stein, MD, PhDSearch for more papers by this authorPublished Online:1 Aug 2019https://doi.org/10.1089/gen.39.08.11AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 39Issue 8Aug 2019 InformationCopyright © by GEN PublishingTo cite this article:Richard A. Stein, MD, PhD.Paper-Based Immunoassays Gain Currency.Genetic Engineering & Biotechnology News.Aug 2019.36-38, 40.http://doi.org/10.1089/gen.39.08.11Published in Volume: 39 Issue 8: August 1, 2019PDF download

Designing Paper-Based Immunoassays for Biomedical Applications.

Paper-based sensors and assays have been highly attractive for numerous biological applications, including rapid diagnostics and assays for disease detection, food safety, and clinical care. In particular, the paper immunoassay has helped drive many applications in global health due to its low cost and simplicity of operation. This review is aimed at examining the fundamentals of the technology, as well as different implementations of paper-based assays and discuss novel strategies for improving their sensitivity, performance, or enabling new capabilities. These innovations can be categorized into using unique nanoparticle materials and structures for detection via different techniques, novel biological species for recognizing biomarkers, or innovative device design and/or architecture.

Detection of resistance protein A (MxA) in paper-based immunoassays with surface enhanced Raman spectroscopy with AuAg nanoshells.

Myxovirus protein A (MxA) is a biomarker that can be used to distinguish between viral and bacterial infections. While MxA lateral flow assays (LFAs) have been successfully used for viral vs. bacterial differential diagnosis for children, the clinically relevant level of MxA for adults has been reported to be 100 times lower, which is too low for traditional LFAs. We present results applying the use of surface enhanced Raman spectroscopy (SERS) to detect MxA. AuAg nanoshells (AuAg NSs) were used to enhance the Raman signal of mercaptobenzoic acid (4-MBA), enabling readout by SERS. The AuAg NSs were conjugated to antibodies for the biomarker of interest, resulting in a "nanotag", that could be used in a dipstick immunoassay for detection. We first optimized the nanotag parameters using anti-human IgG/human IgG as a model antibody/antigen system, and then demonstrated detection of MxA using anti-MxA antibodies. We show that SERS readout of immunoassays for MxA can quantify MxA levels at clinically relevant levels for adult viral infection.

Design Principles for Enhancing Sensitivity in Paper-Based Diagnostics via Large-Volume Processing.

In this work, we characterize the impact of large-volume processing upon the analytical sensitivity of flow-through paper-based immunoassays. Larger sample volumes feature greater molar quantities of available analyte, but the assay design principles which would enable the rapid collection of this dilute target are ill-defined. We developed a finite-element model to explore the operating conditions under which processing large sample volumes via pressure-driven convective flow would yield an improved binding signal. Our simulation results underscore the importance of establishing a high local concentration of the analyte-binding species within the porous substrate. This elevated abundance serves to enhance the binding kinetics, matching the time scale of target capture to the period during which the sample is in contact with the test zone (i.e., the effective residence time). These findings were experimentally validated using the rcSso7d-cellulose-binding domain (CBD) fusion construct, a bifunctional binding protein which adsorbs to cellulose in high abundance. As predicted by our modeling efforts, the local concentration achieved using the rcSso7d-CBD species is uniquely enabling for sensitivity enhancement through large-volume processing. The rapid analyte depletion which occurs at this high surface density also permits the processing of large sample volumes within practical time scales and flow regimes. Using these findings, we present guidance for the optimal means of processing large sample volumes for enhanced assay sensitivity.

Electrochemical Lateral Flow Devices: Towards Rapid Immunomagnetic Assays

AbstractPaper has been used for a long time in the production of low‐cost point‐of‐care (POC) detection devices, most of which rely on the detection of colored bands by the user. However, test strips rarely provide the quantitative detection of biomarkers in clinical samples. In contrast, electrochemical detection enables quantitative detection in paper‐based immunoassays. Here, we demonstrate the production of a simple and economical lateral‐flow electrode device using only screen printing. Contrary to previous work, this device is used to simplify the washing and detection of a short and sensitive immunomagnetic assay for myeloperoxidase (MPO) detection. As we show, after incubation of the sample with magnetic beads (MBs) and detection antibody (Ab) for only 5 min, the whole reaction mixture can be directly pipetted onto the strip. Washing and detection can then be performed, as the MBs are retained with the aid of a magnet, and minimal user intervention is needed. The obtained results reveal that MPO can be detected in 1 : 100 diluted serum with limits of detection and quantification of 0.18 and 0.62 ng mL−1, respectively, in less than 15 min.

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

A paper-based immunoassay to determine HPV vaccination status at the point-of-care

ObjectiveTo develop and evaluate a paper-based point-of-care HPV serology test to determine if an individual has received two or more HPV immunizations. MethodsThe paper-based immunoassay was constructed using a nitrocellulose lateral flow strip with adsorbed HPV16 virus-like particles serving as the capturing moiety. Three capture zones containing virus-like particles were placed in series to allow for visual discrimination between high and low HPV16 plasma antibody concentrations. A plasma separation membrane was used to allow whole blood to be applied directly to the assay. All reagents were dried on glass fiber pads during device fabrication and were rehydrated with buffer at the time of use. A pilot study consisting of 35 subjects with a history of zero, one, two or three HPV vaccines was conducted to evaluate the immunoassay. The completed paper-based immunoassays were scanned for visual interpretation by three researchers who were blinded to the true results and separately evaluated quantitatively using MATLAB. ResultsFor the 28 tests valid for analysis, fifteen subjects reported receiving two or more HPV vaccines, three reported receiving one, and ten reported having no HPV vaccinations. The paper-based immunoassays for all fifteen subjects who reported having received two or more HPV vaccines were judged positive by all researchers. Twelve of the thirteen tests from individuals reporting one or zero vaccinations were deemed negative by all observers. One test from an unvaccinated individual was judged positive by two out of three reviewers. Quantitatively, all tests were correctly separated between the two groups. ConclusionsWe successfully designed and tested a HPV serology test amenable to the point-of-care. The device showed promising results in a pilot study for discriminating between those who received two or more HPV vaccinations and those who did not. Furthermore, this device offers a platform for producing other semi-quantitative point-of-care serological tests.

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.

A device architecture for three-dimensional, patterned paper immunoassays.

Diagnostic assays can provide valuable information about the health status of a patient, which include detection of biomarkers that indicate the presence of an infection, the progression or regression of a disease, and the efficacy of a course of treatment. Critical healthcare decisions must often be made at the point-of-care, far from the infrastructure and diagnostic capabilities of centralized laboratories. There exists an obvious need for diagnostic tools that are designed to address the unique challenges encountered by healthcare workers in limited-resource settings. Paper, a readily-available and inexpensive commodity, is an attractive medium with which to develop diagnostic assays for use in limited-resource settings. In this article, we describe a device architecture to perform immunoassays in patterned paper. These paper-based devices use a combination of lateral and vertical flow to control the wicking of fluid in three-dimensions. We provide guidelines to aid in the design of these devices and we illustrate how patterning can be used to tune the duration and performance of the assay. We demonstrate the use of these paper-based devices by developing a sandwich immunoassay for human chorionic gonadotropin (hCG) in urine, a biomarker of pregnancy. We then directly compare the qualitative and quantitative results of these paper-based immunoassays to commercially available lateral flow tests (i.e., the home pregnancy test). Our results suggest paper-based devices may find broad utility in the development of immunoassays for use at the point-of-care.

Multiplex microfluidic paper-based immunoassay for the diagnosis of hepatitis C virus infection.

Hepatitis C virus (HCV) infection is a serious and rising global healthcare problem. One critical challenge to tackle this disease is the lack of adequate diagnosis. Here, we develop a multiplex microfluidic paper-based immunoassay, as a novel diagnostic approach, to detect human IgG antibody against HCV (anti-HCV). The paper substrate, highly flammable nitrocellulose (NC), is patterned under ambient temperature by craft punch patterning (CPP) to generate multiple test zones. On the basis of superior merits of patterned paper, this new diagnostic approach demonstrates the key novelty to unprecedentedly combine segmented diagnostic assays into a single multiplex test. The generated diagnostic results are not only informative but can be rapidly and cost-effectively delivered. It would significantly transform the clinical pathway for unwitting individuals with HCV infection. This work highlights the promising role of microfluidic paper-based immunoassays in tackling the diagnostic challenge for the HCV pandemic as well as other diseases.

Point-of-care colorimetric detection with a smartphone

Paper-based immunoassays are becoming powerful and low-cost diagnostic tools, especially in resource-limited settings. Inexpensive methods for quantifying these assays have been shown using desktop scanners, which lack portability, and cameras, which suffer from the ever changing ambient light conditions. In this work, we introduce a novel approach of quantifying colors of colorimetric diagnostic assays with a smartphone that allows high accuracy measurements in a wide range of ambient conditions, making it a truly portable system. Instead of directly using the red, green, and blue (RGB) intensities of the color images taken by a smartphone camera, we use chromaticity values to construct calibration curves of analyte concentrations. We demonstrate the high accuracy of this approach in pH measurements with linear response ranges of 1-12. These results are comparable to those reported using a desktop scanner or silicon photodetectors. To make the approach adoptable under different lighting conditions, we developed a calibration technique to compensate for measurement errors due to variability in ambient light. This technique is applicable to a number of common light sources, such as sun light, fluorescent light, or smartphone LED light. Ultimately, the entire approach can be integrated in an "app" to enable one-click reading, making our smartphone based approach operable without any professional training or complex instrumentation.