RESUMO
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.
Assuntos
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.
Assuntos
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.
Assuntos
Insuficiência Cardíaca , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , Humanos , Limite de Detecção , Imunoensaio , Insuficiência Cardíaca/diagnóstico , Biomarcadores/análise , Fenômenos MagnéticosRESUMO
The diagnosis of many diseases requires monitoring of biomarker levels over a period of time instead of assessing their concentration only once. For example, in case of heart failure determination, the levels of N-terminal prohormone brain natriuretic peptide (NT-proBNP) in blood vary so strongly amongst individuals, that the current procedure of one-time measurement in combination with clinical examination does not allow for accurate assessment of disease severity and progression. Our microfluidic biosensor addresses key characteristics of desirable home-tests which include low limits of detection, small sample volume (less than 10 µL), simple detection strategies, and ready-to-go all-dried long-term stable reagents. Here, electrochemically superior silver nanoparticles (AgNP) were dried directly within the microfluidic channel in a matrix of trehalose sugar doped with Na2SO3 as oxygen scavenger. This successfully prevented AgNP oxidation and enabled dry and ready-to-use storage for at least 18 weeks. Based on this, laser-cut flow chips were developed containing all bioassay reagents needed in a ready-to-go dry format. An oxidation-reduction stripping voltammetry strategy was used for highly sensitive quantification of the AgNPs as electrochemical label. This microfluidic biosensor demonstrated limits of detection for NT-proBNP of 0.57 ng mL-1 with a mean error of 6% (n ≥ 3) in undiluted human serum, which is below the clinically relevant cut-off of 1 ng mL-1. This practical approach has the potential to substitute commonly used lateral-flow assays for various biomarkers, as it offers low patient sample volumes hence supporting simple finger-prick strategies well-known also for other electrochemical biosensors, and independence from the notorious variability in fleece fabrication.
Assuntos
Técnicas Biossensoriais , Insuficiência Cardíaca , Nanopartículas Metálicas , Biomarcadores , Humanos , Indicadores e Reagentes , Microfluídica , Peptídeo Natriurético Encefálico , Fragmentos de Peptídeos , PrataRESUMO
Electrochemical immunosensors enable rapid analyte quantification in small sample volumes, and have been demonstrated to provide high sensitivity and selectivity, simple miniaturization, and easy sensor production strategies. As a point-of-care (POC) format, user-friendliness is equally important and most often not combinable with high sensitivity. As such, we demonstrate here that a sequence of metal oxidation and reduction, followed by stripping via differential pulse voltammetry (DPV), provides lowest limits of detection within a 2-min automatic measurement. In exchanging gold nanoparticles (AuNPs), which dominate in the development of POC sensors, with silver nanoparticles (AgNPs), not only better sensitivity was obtained, but more importantly, the assay protocol could be simplified to match POC requirements. Specifically, we studied both nanoparticles as reporter labels in a sandwich immunoassay with the blood protein biomarker NT-proBNP. For both kinds of nanoparticles, the dose-response curves easily covered the ngâmL-1 range. The mean standard deviation of all measurements of 17% (n ≥ 4) and a limit of detection of 26 ngâmL-1 were achieved using AuNPs, but their detection requires addition of HCl, which is impossible in a POC format. In contrast, since AgNPs are electrochemically less stable, they enabled a simplified assay protocol and provided even lower LODs of 4.0 ngâmL-1 in buffer and 4.7 ngâmL-1 in human serum while maintaining the same or even better assay reliability, storage stability, and easy antibody immobilization protocols. Thus, in direct comparison, AgNPs clearly outperform AuNPs in desirable POC electrochemical assays and should gain much more attention in the future development of such biosensors.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Ouro , Humanos , Imunoensaio/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Reprodutibilidade dos Testes , PrataRESUMO
We describe the use of upconverting phosphors (UCPs) as an amplifier of the colorimetric signal in enzymatic dry chemistry test strips for the determination of glucose. The indicator compound used in the strips attenuated both the emission and excitation wavelengths of the upconverting phosphors, thus taking advantage of the nonlinear relationship between the excitation and emission of these particles. By monitoring the emission of the UCPs under 978 nm excitation, we were able to obtain significantly higher signal response (a steeper calibration curve) from the colorimetric assay compared to a reflectance measurement. The limit of detection of the developed method was 2.1-fold lower than the reflectance-based reference method. The new method was able to determine glucose from a sample of water in the range of 5.7 µM to 22 mM, which covers the typical blood glucose range in newborns and adults. In low analyte concentrations we obtained up to 3-fold improvement in the slope of the assay calibration curve. This difference decreased with increasing analyte concentration and the effect was diminished by the highest glucose concentration. However, the analytical sensitivity (the ratio of slope and standard deviation) was practically identical between the two methods because of the higher deviation in the UCP emission signal.