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![Sensitivity enhancement based on isothermal nucleic acid amplification. A Schematic illustration of RCA integrated with LFA for miRNA detection [35]. B Layout of the PDMS/paper hybrid microfluidic device integrated with on-chip LAMP system [51]. C The whole process for STOPCovid, Version 2 (STOPCovid.v2) Test [52]](publication/355201626/figure/fig1/AS:11431281118645656@1675825383652/Sensitivity-enhancement-based-on-isothermal-nucleic-acid-amplification-A-Schematic.png)
Sensitivity enhancement based on isothermal nucleic acid amplification. A Schematic illustration of RCA integrated with LFA for miRNA detection [35]. B Layout of the PDMS/paper hybrid microfluidic device integrated with on-chip LAMP system [51]. C The whole process for STOPCovid, Version 2 (STOPCovid.v2) Test [52]
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![Sensitivity enhancement based on NC membrane size change [92]. A...](publication/355201626/figure/fig5/AS:11431281118610623@1675825386333/Sensitivity-enhancement-based-on-NC-membrane-size-change-92-A-Schematic-of-the-flow_Q320.jpg)
Conventional lateral flow assay (LFA) is typically performed by observing the color changes in the test lines by naked eyes, which achieves considerable commercial success and has a significant impact on the fields of food safety, environment monitoring, disease diagnosis, and other applications. However, this qualitative detection method is not ve...
Citations
... LFIAs, founded on an affinity-capture-migration technology, occasionally yield falsenegative or false-positive misdiagnoses, yet they continue to be used as a screening tool [100][101][102][103]. Conversely, immunoaffinity capillary electrophoresis (IACE), based on the principle of an affinity-capture-separation or a two-dimensional technology, is a popular test due to its ability to confirm results [70,[103][104][105][106][107][108]. ...
Many ailments can be diagnosed while they are asymptomatic, meaning that the patient has no signs or symptoms of a progressing disease. If caught in their initial stage of formation, these disorders can be effectively treated, leading to successful outcomes; curative therapies can halt illnesses from advancing, thus improving the quality of life and long-term survival of the patient. Still, cutting-edge upgrades in precision technologies are necessary for early, reliable, affordable, and rapid disease detection, but also vital for the well-being of people and the future of global public health. The emerging role and utility of non-invasive and repeatable diagnostic test approaches for the detection of health conditions have been exemplified by liquid biopsies based on genomic biomarkers. As such, biological fluids permit any measurable molecular indicator or signature (e.g., proteins) to provide valuable information on an individual's wellness and/or disease. Among the bodily secretions used for non-invasive diagnostics is sputum, a complex viscous gel-like biopolymeric network that has gained growing recognition as a rich source of biomarkers of airway infections and pulmonary diseases, and serves as a determinant to reveal other illnesses. As per the World Health Organization, the burden of respiratory conditions is exacerbated by factors ranging from considerable subjection to air pollution and occupational contaminants to tobacco smoking and secondhand smoke, in addition to poor socioeconomic status. Due to the likely increase in these determinants, respiratory tract ailments are on the rise, affecting the health of many individuals, in addition to putting stress on healthcare facilities and services worldwide. The aim of this study was to perform a narrative review of sputum constituents with an emphasis on proteins and glycoproteins assessed as possible biomarkers of lung and other organ diseases. A search was conducted using mucus, sputum proteomics, sputum biomarkers, and point-of-care testing as keywords employing Google, PubMed (MEDLINE), and Web of Science, selecting the most referenced and related papers of the last decade. We, therefore, highlight the need to use expectorated or induced sputum specimens as a routine sample source for testing valuable protein biomarkers to diagnose these chronic disorders, predict inflammation and disease progression, as well as monitor the effectiveness of treatments. Further, we discuss the urgent need for fast and reliable point-of-care methods to detect and quantify crucial protein biomarkers in sputum specimens, and the limitations faced when dealing with their complex matrices.
... LFIAs, founded on an affinity-capture-migration technology occasionally yield false-negative or false-positive misdiagnoses, yet they continue to be used as a screening tool [92][93][94][95]. Conversely, immunoaffinity capillary electrophoresis (IACE), based on the principle of an affinity-captureseparation or a two-dimensional technology, are popular tests due to their ability to confirm results [62,[95][96][97][98][99][100]. ...
Many ailments can be diagnosed while they are asymptomatic, meaning that the patient has no signs or symptoms of a progressing disease. If caught in their initial stage of formation, these maladies can be effectively treated, leading to successful outcomes; curative therapies can halt illnesses from advancing, thus improving the quality of life, and long-term survival of the patient. Still cutting-edge upgrades in precision technologies are necessary for early, reliable, affordable, and rapid disease detection, but also are vital for the well-being of people and the future of global public health. The emerging role and utility of non-invasive and repeatable diagnostic test approaches for detection of health conditions has been liquid biopsies based on genomic biomarkers. As such, biological fluids permit any measurable molecular indicator or signature (e.g., proteins), to provide valuable information on individual’s wellness and/or disease. Among the bodily secretions used for non-invasive diagnostics is sputum, a complex viscous gel-like biopolymeric network, that has gained growing recognition as a rich source of biomarkers of airway infections, pulmonary diseases, and serves as a determinant to reveal other illnesses. As per the World Health Organization, the burden of respiratory conditions is exacerbated by factors, ranging from considerable subjection to air pollution and occupational contaminants, to tobacco smoking and second-hand smoke, in addition to poor socio-economic status. Due to the likely increase of these determinants, respiratory tract ailments are on the rise, affecting the health of many individuals, in addition to putting stress on healthcare facilities and services worldwide. We therefore highlight the need to use expectorated or induced sputum specimens as a routine sample source for testing valuable protein biomarkers to diagnose these chronic maladies, to predict inflammation and disease progression, as well as monitor the effectiveness of treatments. Further, we discuss the urgency for fast and reliable point-of-care methods to detect and quantify crucial protein biomarkers in sputum specimens, and limitations faced when dealing with their complex matrices.
... 1,14,15 To overcome these analytical challenges, a range of strategies for improvement have been extensively investigated in the last years. 16 A number of reviews have exclusively focused on the increase of assay sensitivity and mention the (i) improvements of pad geometries, 17 (ii) controlling the flow speed, 1,18,19 and (iii) engineering novel detection labels, 16 among others. In addition to the variety of reviews, more than 2000 articles about LFAs were published in the past decade. ...
Fibrinogen is a coagulation factor in human blood and the first one to reach critical levels in major bleeding. Hypofibrinogenemia (a too low fibrinogen concentration in blood) poses great challenges to first responders, clinicians, and healthcare providers since it represents a risk factor for exsanguination and massive transfusion requirements. Thus, the rapid assessment of the fibrinogen concentration at the point of care has gained considerable importance in preventing and managing major blood loss. However, in whole blood measurements, hematocrit variations affect the amount (volume fraction) of plasma that passes the detection zone. In an attempt to accurately determine realistic critical levels of fibrinogen (<1.5 mg/mL) in patients needing immediate treatment and medical interventions, we have developed novel diagnostic systems capable of estimating hematocrit and critical fibrinogen concentrations. A lateral flow assay (LFA) for the detection of fibrinogen has been developed by establishing a workflow employing rapid characterization methods to streamline LFA development. The integration of two detection lines enables (i) the identification of fibrinogen (first line) present in the sample and (ii) the determination of the clinically critical fibrinogen concentrations below 1.5 mg/mL (second line). Furthermore, the paper-based separation of blood cells from plasma provides a semiquantitative estimate of the hematocrit by analyzing the fractions. Initial validation of the point-of-care (PoC) hematocrit test revealed good comparability to a standard laboratory method. The developed diagnostic systems have the ability to accelerate decision-making in cases with major bleeding.
... In addition, the sensitivity and specificity of these assembled devices depends partly on the concentration density of antibodies and control antigens immobilized in the test line. [9] When compared to gold standard laboratory assays, such as csELISA, current rapid assays are less sensitive and accurate and need higher concentrations of the antigen in the samples tested. [3,10] The use of naturally abundant, low-cost materials to locally develop LFAs and DSAs could increase equity in the availability of rapid testing tools for malaria transmission and control monitoring. ...
Enhanced and rapid surveillance for diseases is critical to public health and meeting United Nations' Sustainable Development Goal for Good Health and Well-being by allowing for targeted and accelerated prevention and control response strategies. Human malaria, caused by Plasmodium spp. and transmitted by mosquitoes is no exception. Advances in sustainable materials provide an opportunity to improve fast, sustainable, and equitable testing assays. Here, naturally abundant polymers and biomaterials, such as cellulose nanocrystals (CNCs) and chitosan, were used to increase antibody density deposition on the assay detection line when compared to traditional free antibody deposition, and thus the sensitivity, of easily assembled rapid tests designed to detect Plasmod-ium vivax infective (sporozoite) parasites in mosquitoes, a critical indicator of malaria transmission. The immobilization of antibodies onto chitosan-coated CNCs allowed for antigen detection with a lower number of antibodies used in each test; likewise, the immobilization allowed to directly place the CNC-Ab without the traditionally needed blockers layer on the paper like bovine serum albumin (BSA). This bio-based prototype of a paper-based dipstick assay shows a promising pathway for the development of rapid disease surveillance tools using sustainable and globally available materials. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
... Many efforts have been made to increase the sensitivity of LFIA, which are summarized in multiple reviews [3,7,18,24,25]. However, most reviews examine all the existing approaches for LOD reduction, from selecting high-affine binders to instrumental methods of signal registration. ...
Lateral flow immunoassay (LFIA) has found a broad application for testing in point-of-care (POC) settings. LFIA is performed using test strips—fully integrated multimembrane assemblies containing all reagents for assay performance. Migration of liquid sample along the test strip initiates the formation of labeled immunocomplexes, which are detected visually or instrumentally. The tradeoff of LFIA’s rapidity and user-friendliness is its relatively low sensitivity (high limit of detection), which restricts its applicability for detecting low-abundant targets. An increase in LFIA’s sensitivity has attracted many efforts and is often considered one of the primary directions in developing immunochemical POC assays. Post-assay enhancements based on chemical reactions facilitate high sensitivity. In this critical review, we explain the performance of post-assay chemical enhancements, discuss their advantages, limitations, compared limit of detection (LOD) improvements, and required time for the enhancement procedures. We raise concerns about the performance of enhanced LFIA and discuss the bottlenecks in the existing experiments. Finally, we suggest the experimental workflow for step-by-step development and validation of enhanced LFIA. This review summarizes the state-of-art of LFIA with chemical enhancement, offers ways to overcome existing limitations, and discusses future outlooks for highly sensitive testing in POC conditions.
... Sena-Torralba et al. discussed how nanomaterial could address the inherent weaknesses of traditional LFAs and summarized the obstacles toward commercialization and the creation of new hardware as well as a portable reader for LFA quantification and analysis [79]. Deng et al. reviewed the evolving methods to boost the sensitivity of LFAs, and the future viewpoints and limitations in this research area were also discussed [80]. Liu et al. exhaustively discussed approaches for augmenting the sensitivity and specificity of LFAs [69]. ...
Lateral flow tests are one of the most important types of paper-based point-of-care (POCT) diagnostic tools. It shows great potential as an implement for improving the rapid screening and management of infections in global pandemics or other potential health disorders by using minimally expert staff in locations where no sophisticated laboratory services are accessible. They can detect different types of biomarkers in various biological samples and provide the results in a little time at a low price. An important challenge regarding conventional LFAs is increasing their sensitivity and specificity. There are two main approaches to increase sensitivity and specificity, including assay improvement and target enrichment. Assay improvement comprises the assay optimization and signal amplification techniques. In this study, a summarize of various sensitivity and specificity enhancement strategies with an objective evaluation are presented, such as detection element immobilization, capillary flow rate adjusting, label evolution, sample extraction and enrichment, etc. and also the key findings in improving the LFA performance and solving their limitations are discussed along with numerous examples.
... Therefore, rapid, multiplexed genotypic assays are required for CRE testing. Lateral flow test strips (LFTS) are highly desirable platforms for rapid diagnostic systems due to their simplicity, speed, affordability, and user-friendliness [12,13]. With the emergence of newer design strategies and advanced multiplexed testing techniques, LFTS sensors have progressed from single-analyte detections to versatile multiplexing analyses [14]. ...
Carbapenem-resistant Enterobacterales pose significant global health challenges due to their rapid spread and ability to hydrolyse various beta-lactam antibiotics. Rapid tests for these carbapenemase genes are crucial to ensure appropriate prescription administration and infection control. In this study, we developed a rapid visual nanodiagnostic platform for multiplexed detection of carbapenemase genes using a lateral flow strip. The nanodiagnostic strip was designed with separate barcoded DNA tetrahedrons for the blaKPC and blaNDM genes. These tetrahedrons were distributed on a nitrocellulose membrane at two different test lines as capture probes. When tested against a panel of carbapenemase genes, the tetrahedral probes captured single-stranded amplicons of asymmetric PCR via strand hybridisation. The amplicons acted as bridging elements, binding the DNA-modified gold nanoparticles to the test line of the strip, resulting in clear visual readouts specific to the blaKPC and blaNDM genes. By employing barcoded tetrahedrons and asymmetric PCR in conjunction with the lateral flow strip, a single diagnostic test enabled the detection of multiple carbapenemase genes. The test yielded results as low as 0.12 fM for blaKPC and 0.05 fM for blaNDM within 75 min. Furthermore, the strip effectively identified specific carbapenemase genes in clinical isolates using real-time PCR, antibody-based lateral flow systems for carbapenemase detection, and carbapenemase phenotype experiments. Thus, the strip develop has a high potential for testing blaKPC and blaNDM genes in practice.
Graphical Abstract
... Surface-enhanced Raman spectroscopy scattering (SERS) originates from the presence of a strong local plasma electromagnetic field on a rough surface at the nanoscale, and when molecules are adsorbed on the surface of a metal structure their scattering cross section is dramatically amplified by the local electromagnetic field of the metal surface, resulting in a 10 10 to 10 11 fold increase in the Raman intensity of the molecules (Camden et al., 2008). Compared with gold nanoparticlebased colorimetric methods, the SERS immunochromatographic technique takes advantage of the high sensitivity and accuracy of SERS, allowing for a sensitivity increase of typically 3 to 4 orders of magnitude (Wang et al., 2017;Deng et al., 2021). SERS method is often combined with Au or Ag (Sultangaziyev et al., 2022). ...
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is still in an epidemic situation, which poses a serious threat to the safety of people and property. Rapid diagnosis and isolation of infected individuals are one of the important methods to control virus transmission. Existing lateral flow immunoassay techniques have the advantages of rapid, sensitive, and easy operation, and some new options have emerged with the continuous development of nanotechnology. Such as lateral flow immunoassay test strips based on colorimetric-fluorescent dual-mode and gold nanoparticles, Surface Enhanced Raman Scattering, etc., these technologies have played an important role in the rapid diagnosis of COVID-19. In this paper, we summarize the current research progress of lateral flow immunoassay in the field of Severe Acute Respiratory Syndrome Coronavirus 2 infection diagnosis, analyze the performance of Severe Acute Respiratory Syndrome Coronavirus 2 lateral flow immunoassay products, review the advantages and limitations of different detection methods and markers, and then explore the competitive CRISPR-based nucleic acid chromatography detection method. This method combines the advantages of gene editing and lateral flow immunoassay and can achieve rapid and highly sensitive lateral flow immunoassay detection of target nucleic acids, which is expected to be the most representative method for community and clinical point-of-care testing. We hope that researchers will be inspired by this review and strive to solve the problems in the design of highly sensitive targets, the selection of detection methods, and the enhancement of CRISPR technology, to truly achieve rapid, sensitive, convenient, and specific detection of novel coronaviruses, thus promoting the development of novel coronavirus diagnosis and contributing our modest contribution to the world’s fight against epidemics.
... Promising examples include isothermal nucleic acid amplification such as the loop-mediated isothermal amplification for the detection of malaria 29 or protein preconcentration. 30 Yet, simplification of these processes or automation has not been addressed by the authors. Sequential delivery of the reagents was found by Liang et al. to improve the sensitivity. ...
Point-of-care (POC) diagnostics in particular focuses on the timely identification of harmful conditions close to the patients' needs. For future healthcare these diagnostics could be an invaluable tool especially in a digitalized or telemedicine-based system. However, while paper-based POC tests, with the most prominent example being the lateral flow assay (LFA), have been especially successful due to their simplicity and timely response, the COVID-19 pandemic highlighted their limitations, such as low sensitivity and ambiguous responses. This perspective discusses strategies that are currently being pursued to evolve such paper-based POC tests toward a superior diagnostic tool that provides high sensitivities, objective result interpretation, and multiplexing options. Here, we pinpoint the challenges with respect to (i) measurability and (ii) public applicability, exemplified with select cases. Furthermore, we highlight promising endeavors focused on (iii) increasing the sensitivity, (iv) multiplexing capability, and (v) objective evaluation to also ready the technology for integration with machine learning into digital diagnostics and telemedicine. The status quo in academic research and industry is outlined, and the likely highly relevant role of paper-based POC tests in future healthcare is suggested.
... Due to these reasons, ICA is less sensitive, often significantly, than laboratory immunoassay techniques, such as microplate enzyme immunoassay. Therefore, new variants of ICA with higher sensitivity are in demand [4][5][6]. Such improved techniques make the detection of very low concentrations of analytes possible, thereby enabling the performance of point-of-care testing for compounds with low levels in tested samples. ...
The work considers a combination of three enhancing approaches for immunochromatographic assay (ICA) and the integration of their impacts into changes of the limit of detection (LOD). Human fatty acid binding protein (FABP), an early biomarker of acute myocardial infarction, was the target analyte. Starting from the common ICA protocol with an LOD equal to 11.2 ng/mL, three approaches were realized: (1) replacement of spherical gold nanoparticles with gold nanoflowers having a branched surface (20-fold lowering the LOD); (2) enhanced labeling of immune complexes via nanoparticle aggregates (15-fold lowering); (3) in-situ growth of bound nanoparticles by reduction of gold salts (3-fold lowering). Single and combined implementations of these approaches have been studied. It has been shown that the LOD decrease for combined approaches is close to the multiplied contribution of each of them. The final LOD for FABP was 0.05 ng/mL, which is 220 times lower than the LOD for the common ICA protocol. The efficiency of the enhanced ICA with three combined approaches was confirmed by testing human serum samples for FABP presence and content. The development presents a new efficient technique for rapid sensitive detection of FABP for medical diagnostics. Moreover, the demonstrated multiple enhancements could be applied for various demanded analytes.
