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Through enabling whole blood detection in point-of-care testing (POCT), sedimentation-based plasma separation promises to enhance the functionality and extend the application range of lateral flow assays (LFAs). To streamline the entire process from the introduction of the blood sample to the generation of quantitative immune-fluorescence results, we combined a simple plasma separation technique, an immunoreaction, and a micropump-driven external suction control system in a polymer channel-based LFA. Our primary objective was to eliminate the reliance on sample-absorbing separation membranes, the use of active separation forces commonly found in POCT, and ultimately allowing finger prick testing. Combining the principle of agglutination of red blood cells with an on-device sedimentation-based separation, our device allows for the efficient and fast separation of plasma from a 25-µL blood volume within a mere 10 min and overcomes limitations such as clogging, analyte adsorption, and blood pre-dilution. To simplify this process, we stored the agglutination agent in a dried state on the test and incorporated a filter trench to initiate sedimentation-based separation. The separated plasma was then moved to the integrated mixing area, initiating the immunoreaction by rehydration of probe-specific fluorophore-conjugated antibodies. The biotinylated immune complex was subsequently trapped in the streptavidin-rich detection zone and quantitatively analyzed using a fluorescence microscope. Normalized to the centrifugation-based separation, our device demonstrated high separation efficiency of 96% and a yield of 7.23 µL (= 72%). Furthermore, we elaborate on its user-friendly nature and demonstrate its proof-of-concept through an all-dried ready-to-go NT-proBNP lateral flow immunoassay with clinical blood samples.
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Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Humanos , Peptídeo Natriurético Encefálico/sangue , Peptídeo Natriurético Encefálico/isolamento & purificação , Fragmentos de Peptídeos/sangue , Testes Imediatos , Imunoensaio/métodos , Imunoensaio/instrumentação , Desenho de EquipamentoRESUMO
Membrane-based lateral flow immunoassays (LFAs) have been employed as early point-of-care (POC) testing tools in clinical settings. However, the varying membrane properties, uncontrollable sample transport in LFAs, visual readout, and required large sample volumes have been major limiting factors in realizing needed sensitivity and desirable precise quantification. Addressing these challenges, we designed a membrane-free system in which the desirable three-dimensional (3D) structure of the detection zone is imitated and used a small pump for fluid flow and fluorescence as readout, all the while maintaining a one-step assay protocol. A hydrogel-like protein-polyelectrolyte complex (PPC) within a polyelectrolyte multilayer (PEM) was developed as the test line by complexing polystreptavidin (pSA) with poly(diallyldimethylammonium chloride) (PDDA), which in turn was layered with poly(acrylic acid) (PAA) resulting in a superior 3D streptavidin-rich test line. Since the remainder of the microchannel remains material-free, good flow control is achieved, and with the total volume of 20 µL, 7.5-fold smaller sample volumes can be used in comparison to conventional LFAs. High sensitivity with desirable reproducibility and a 20 min total assay time were achieved for the detection of NT-proBNP in plasma with a dynamic range of 60-9000 pg·mL-1 and a limit of detection of 56 pg·mL-1 using probe antibody-modified fluorescence nanoparticles. While instrument-free visual detection is no longer possible, the developed lateral flow channel platform has the potential to dramatically expand the LFA applicability, as it overcomes the limitations of membrane-based immunoassays, ultimately improving the accuracy and reducing the sample volume so that finger-prick analyses can easily be done in a one-step assay for analytes present at very low concentrations.
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Biomarcadores , Compostos de Amônio Quaternário , Humanos , Imunoensaio/métodos , Biomarcadores/análise , Biomarcadores/sangue , Peptídeo Natriurético Encefálico/sangue , Peptídeo Natriurético Encefálico/análise , Limite de Detecção , Resinas Acrílicas/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/sangue , Polietilenos/química , Poliestirenos/químicaRESUMO
Point-of-care sensors targeting blood marker analysis must be designed to function with very small volumes since acquiring a blood sample through a simple, mostly pain-free finger prick dramatically limits the sample size and comforts the patient. Therefore, we explored the potential of converting a conventional lateral flow assay (LFA) for a significant biomarker into a self-contained and compact polymer channel-based LFA to minimize the sample volume while maintaining the analytical merits. Our primary objective was to eliminate the use of sample-absorbing fleece and membrane materials commonly present in LFAs. Simultaneously, we concentrated on developing a ready-to-deploy one-step LFA format, characterized by dried reagents, facilitating automation and precise sample transport through a pump control system. We targeted the detection of the heart failure biomarker NT-proBNP in only 15 µL human whole blood and therefore implemented strategies that ensure highly sensitive detection. The biosensor combines streptavidin-functionalized magnetic beads (MNPs) as a 3D detection zone and fluorescence nanoparticles as signal labels in a sandwich-based immunoassay. Compared to the currently commercialized LFA, our biosensor demonstrates comparable analytical performance with only a tenth of the sample volume. With a detection limit of 43.1 pgâmL-1 and a mean error of 18% (n ≥ 3), the biosensor offers high sensitivity and accuracy. The integration of all-dried long-term stable reagents further enhances the convenience and stability of the biosensor. This lateral flow channel platform represents a promising advancement in point-of-care diagnostics for heart failure biomarkers, offering a user-friendly and sensitive platform for rapid and reliable testing with low finger-prick blood sample volumes.
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Insuficiência Cardíaca , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Humanos , Limite de Detecção , Imunoensaio , Insuficiência Cardíaca/diagnóstico , Biomarcadores/análise , Fenômenos MagnéticosRESUMO
Multiplexing is a relevant strategy for biosensors to improve accuracy and decision-making due to the increased amount of simultaneously obtained information. Liposomes offer unique benefits for label-based multiplexing since a variety of different marker molecules can be encapsulated, leading to intrinsic signal amplification and enabling a variety of detection formats. We successfully developed an electrochemical (EC) liposome-based platform technology for the simultaneous detection of at least three analytes by studying parameters to ensure specific and sensitive bioassay performance. Influenza A and B and SARS-CoV-2 sequences served as model system in a standard sandwich hybridization assay. Studies included encapsulants, probe distribution on liposomes and capture beads, assay setup and interferences between liposomes to also ensure a generalization of the platform. Ruthenium hexamine(III), potassium hexacyanoferrate(II) and m-carboxy luminol, when encapsulated separately into a liposome, provided desirable long-term stability of at least 12 months and no cross-signals between liposomes. Through the optimization process, low limits of detections of 1.6 nmol L-1, 125 pmol L-1 and 130 pmol L-1, respectively, were achieved in a multiplexed assay setup, which were similar to singleplex assays. Non-specific interactions were limited to 25.1%, 7.6% and 7.5%, respectively, through sequential liposome incubations and singleplex capture bead designs. Here, ruthenium hexamine liposomes had only mediocre performance so that low overall signal strength translated into higher LODs and worse specificity. A different marker such as ferroin may be an option in the future. The identification of further electrochemical markers will provide new opportunities for liposomes to function as multiplex, orthogonal or internal standard labels in electrochemical bioassays.
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Técnicas Eletroquímicas , Vírus da Influenza B , Limite de Detecção , Lipossomos , SARS-CoV-2 , Lipossomos/química , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , Técnicas Eletroquímicas/métodos , Humanos , Vírus da Influenza B/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Técnicas Biossensoriais/métodos , Influenza Humana/diagnóstico , Influenza Humana/virologia , COVID-19/diagnóstico , COVID-19/virologiaRESUMO
Laser-induced graphene (LIG) has received much attention in recent years as a possible transducer material for electroanalytical sensors. Its simplicity of fabrication and good electrochemical performance are typically highlighted. However, we found that unmodified and untreated LIG electrodes had a limited shelf-life for certain electroanalytical applications, likely due to the adsorption of adventitious hydrocarbons from the storage environment. Electrode responses did not change immediately after exposure to ambient conditions but over longer periods of time, probably due to the immense specific surface area of the LIG material. LIG shelf-life is seldomly discussed prominently in the literature, yet overall trends for solutions to this challenge can be identified. Such findings from the literature regarding the long-term storage stability of LIG electrodes, pure and modified, are discussed here along with explanations for likely protective mechanisms. Specifically, applying a protective coating on LIG electrodes after manufacture is possibly the easiest method to preserve electrode functionality and should be identified as a trend for well-performing LIG electrodes in the future. Furthermore, suggested influences of the accompanying LIG microstructure/morphology on electrode characteristics are evaluated.
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Point-of-care (POC) devices are rapid, simple, portable, inexpensive, and convenient, but typically they only deliver qualitative results when used in the form of a lateral flow assay (LFA). Electrochemical detection could improve their sensitivity and ensure quantitative detection; however, a breakthrough in material-based technology is needed. We demonstrate a new concept in which electrodes are directly embedded within the lateral flow, enabling flow-through and hence interaction with the entire sample. This is accomplished through laser-induced carbon nanofibers (LCNFs) made by electrospinning Matrimid into nanofiber mats with subsequent pyrolyzing of electrode structures through a CO2 laser. Their highly porous 3D structure and superior graphene-like electrochemical properties are ideally suited for flow-through electrochemical LFA (EC-LFA), where the LCNFs are simply added in line with the other membranes. After optimization of the setup, biological binding assays typical for LFA diagnostics were successfully implemented, enabling the highly sensitive and quantitative detection of 137 pM DNA target sequences of a pathogenic organism that rivals the performance of pump-controlled microfluidic bioassays. This demonstrates that LCNF-based transducers can transform paper-based diagnostic tests to enable precise, quantitative analysis without reliance on cost-intensive read-out systems.
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The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) in 2019 caused an increased interest in neutralizing antibody tests to determine the immune status of the population. Standard live-virus-based neutralization assays such as plaque-reduction assays or pseudovirus neutralization tests cannot be adapted to the point-of-care (POC). Accordingly, tests quantifying competitive binding inhibition of the angiotensin-converting enzyme 2 (ACE2) receptor to the receptor-binding domain (RBD) of SARS-CoV-2 by neutralizing antibodies have been developed. Here, we present a new platform using sulforhodamine B encapsulating liposomes decorated with RBD as foundation for the development of both a fluorescent, highly feasible high-throughput (HTS) and a POC-ready neutralizing antibody assay. RBD-conjugated liposomes are incubated with serum and subsequently immobilized in an ACE2-coated plate or mixed with biotinylated ACE2 and used in test strip with streptavidin test line, respectively. Polyclonal neutralizing human antibodies were shown to cause complete binding inhibition, while S309 and CR3022 human monoclonal antibodies only caused partial inhibition, proving the functionality of the assay. Both formats, the HTS and POC assay, were then tested using 20 sera containing varying titers of neutralizing antibodies, and a control panel of sera including prepandemic sera and reconvalescent sera from respiratory infections other than SARS-CoV-2. Both assays correlated well with a standard pseudovirus neutralization test (r = 0.847 for HTS and r = 0.614 for POC format). Furthermore, excellent correlation (r = 0.868) between HTS and POC formats was observed. The flexibility afforded by liposomes as signaling agents using different dyes and sizes can hence be utilized in the future for a broad range of multianalyte neutralizing antibody diagnostics.
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COVID-19 , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2 , Lipossomos , Anticorpos Antivirais , Sistemas Automatizados de Assistência Junto ao Leito , COVID-19/diagnóstico , Anticorpos NeutralizantesRESUMO
Silver nanoparticles (AgNPs) have long been overshadowed by gold NPs' success in sensor and point-of-care (POC) applications. However, their unique physical, (electro)chemical, and optical properties make them excellently suited for such use, as long as their inherent higher instability toward oxidation is controlled. Recent advances in this field provide novel strategies that demonstrate that the AgNPs' inherent capabilities improve sensor performance and enable the specific detection of analytes at low concentrations. We provide an overview of these advances by focusing on the nanosized Ag (in the range of 1-100 nm) properties with emphasis on optical and electrochemical biosensors. Furthermore, we critically assess their potential for point-of-care sensors discussing advantages as well as limitations for each detection technique. We can conclude that, indeed, strategies using AgNP are ready for sensitive POC applications; however, research focusing on the simplification of assay procedures is direly needed for AgNPs to make the successful jump into actual applications.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Ressonância de Plasmônio de Superfície/métodos , Prata/química , Nanopartículas Metálicas/química , Sistemas Automatizados de Assistência Junto ao Leito , Técnicas Biossensoriais/métodosRESUMO
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.
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Sensors, ranging from in vivo through to single-use systems, employ protective membranes or hydrogels to enhance sample collection or serve as filters, to immobilize or entrap probes or receptors, or to stabilize and enhance a sensor's lifetime. Furthermore, many applications demand specific requirements such as biocompatibility and non-fouling properties for in vivo applications, or fast and inexpensive mass production capabilities for single-use sensors. We critically evaluated how membrane materials and their deposition methods impact optical and electrochemical systems with special focus on analytical figures of merit and potential toward large-scale production. With some chosen examples, we highlight the fact that often a sensor's performance relies heavily on the deposition method, even though other methods or materials could in fact improve the sensor. Over the course of the last 5 years, most sensing applications within healthcare diagnostics included glucose, lactate, uric acid, O2, H+ ions, and many specific metabolites and markers. In the case of food safety and environmental monitoring, the choice of analytes was much more comprehensive regarding a variety of natural and synthetic toxicants like bacteria, pesticides, or pollutants and other relevant substances. We conclude that more attention must be paid toward deposition techniques as these may in the end become a major hurdle in a sensor's likelihood of moving from an academic lab into a real-world product.
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Poluentes Ambientais , Hidrogéis , Polímeros , Glucose , BactériasRESUMO
3D-carbon nanomaterials have proven to be high-performance transducers in electrochemical sensors but their integration into miniaturized devices is challenging. Herein, we develop printable freestanding laser-induced carbon nanofibers (f-LCNFs) with outstanding analytical performance that furthermore can easily allow such miniaturization through a paper-based microfluidic strategy. The f-LCNF electrodes were generated from electrospun polyimide nanofibers and one-step laser carbonization. A three-electrode system made of f-LCNFs exhibited a limit of detection (LOD) as low as 1 nM (S/N = 8) for anodic stripping analysis of silver ions, exhibiting the peak at ca. 100 mV vs f-LCNFs RE, without the need of stirring. The as-described system was implemented in miniaturized devices via wax-based printing, in which their electroanalytical performance was characterized for both outer- and inner-sphere redox markers and then applied to the detection of dopamine (the peak appeared at ca. 200 mV vs f-LCNFs RE) with a remarkable LOD of 55 pM. When modified with Nafion, the f-LCNFs were highly selective to dopamine even against high concentrations of uric and ascorbic acids. Especially the integration into closed microfluidic systems highlights the strength 3D porous structures provides excellent analytical performance paving the way for their translation to affordable lab-on-a-chip devices where mass-production capability, unsophisticated fabrication techniques, transfer-free, and customized electrode designs can be realized.
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Carbono , Nanofibras , Carbono/química , Nanofibras/química , Técnicas Eletroquímicas/métodos , Dopamina/análise , Prata , TransdutoresRESUMO
After years of disappointing clinical results, the tide has finally changed and complement targeted-therapies have become a validated and accepted treatment option for several diseases. These accomplishments have revitalized the field and brought renewed attention to the prospects that complement therapeutics can offer. Streamlining diagnostics and therapeutics is imperative in this new era of clinical use of complement therapeutics. However, the incredible success in therapeutics has not been accompanied by the development of novel standardized tools for complement testing. Complement biomarkers can assist in the risk assessment and diagnosis of diseases as well as the prediction of disease progression and treatment response. Recently, a group of complement proteins has been suggested to be highly relevant in various complement-associated disorders, namely the human factor H (FH) protein family. This family of closely related proteins consists of FH, FH-like protein 1, and five factor H-related proteins, and they have been linked to eye, kidney, infectious, vascular, and autoimmune diseases as well as cancer. The goal of this review is to provide a comprehensive overview of the available data on circulating levels of FH and its related proteins in different pathologies. In addition, we examined the current literature to determine the clinical utility of measuring levels of the FH protein family in health and disease. Finally, we discuss future steps that are needed to make their clinical translation a reality.
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Fator H do Complemento , Proteínas do Sistema Complemento , Progressão da Doença , Humanos , Rim/metabolismoRESUMO
High performance, laser-induced graphene (LIG) electrodes were integrated into adhesive tape-based microfluidic channels to realize both electrochemical (EC) and electrochemiluminescent (ECL) detection approaches. This provides strategies for low limits of detection, simple hardware requirements and inexpensive fabrication, which are characteristics required for assays in the competitive point-of-care (POC) sensor field. Here, electrode design and microchannel dimensions were studied and a DNA hybridization assay with liposomes for signal amplification was developed for the specific detection of DNA derived from Cryptosporidium parvum as the model analyte. Liposomes entrapped either Ru(bpy)32+ or K4[Fe(CN)6] generating ECL- and EC-signal amplification, respectively. This new microchip provided all desirable analytical figures of merit needed for POC applications. Specifically, a desirable one-step assay was designed which provided a limit of detection of 3 pmol L-1 for the ECL and 47 pmol L-1 for the EC approach and furthermore enabled highly specific detection considering that at room temperature in this simple setup a single nucleotide polymorphism resulted in a signal decrease of 58%, whereas a decrease of > 98% was observed for non-matching sequences present in 10-fold excess. Direct detection in various matrices ranging from drinking water to soil extracts was also achieved. It is concluded that the simple and inexpensive fabrication in combination with signal amplification strategies makes these concepts relevant for on-site pathogen detection in resource-limited environments.
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Técnicas Biossensoriais , Criptosporidiose , Cryptosporidium , Água Potável , Grafite , Compostos Organometálicos , Técnicas Biossensoriais/métodos , DNA , Técnicas Eletroquímicas/métodos , Eletrodos , Humanos , Lasers , Lipossomos , Medições Luminescentes/métodos , Microfluídica , SoloRESUMO
[This corrects the article DOI: 10.3389/fimmu.2021.660194.].
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Gold nanoparticle-catalyzed chemiluminescence (CL) of luminol is an attractive alternative to strategies relying on enzymes, as their aggregation leads to significantly enhanced CL signals. Consequently, analytes disturbing such aggregation will lead to an easy-to-quantify weakening of the signal. Based on this concept, a homogeneous aptamer-based assay for the detection of sulfadimethoxine (SDM) has been developed as a microfluidic CL flow-injection platform. Here, the efficient mixing of gold nanoparticles, aptamers, and analyte in short channel distances is of utmost importance, and two-dimensional (2D) and three-dimensional (3D) mixer designs made via Xurography were investigated. In the end, since 2D designs could not provide sufficient mixing, a laminated 3D 5-layer microfluidic mixer was developed and optimized with respect to mixing capability and observation by the charge-coupled device (CCD) camera. Furthermore, the performance of standard luminol and its more hydrophilic derivative m-carboxy luminol was studied identifying the hydrophilic derivative to provide tenfold more signal enhancement and reliable results. Finally, the novel detection platform was used for the specific detection of SDM via its aptamer and yielded a stunning dynamic range over 5 orders of magnitude (0.01-1000 ng/ml) and a limit of detection of 4 pg/ml. This new detection concept not only outperforms other methods for SDM detection, but can be suggested as a new flow-injection strategy for aptamer-based rapid and cost-efficient analysis in environmental monitoring and food safety.