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Dot-blot immunoassays are widely used for the user-friendly detection of clinical biomarkers. However, the majority of dot-blot assays have only limited sensitivity and are only used for qualitative or semiquantitative analysis. To overcome this limitation, we have employed labels based on photon-upconversion nanoparticles (UCNPs) that exhibit anti-Stokes luminescence and can be detected without optical background interference. First, the dot-blot immunoassay on a nitrocellulose membrane was optimized for the quantitative analysis of human serum albumin (HSA), resulting in a limit of detection (LOD) of 0.19 ng/mL and a signal-to-background ratio (S/B) of 722. Commercial quantum dots were used as a reference label, reaching the LOD of 4.32 ng/mL and the S/B of 3, clearly indicating the advantages of UCNPs. In addition, the potential of UCNP-based dot-blot for real sample analysis was confirmed by analyzing spiked urine samples, reaching the LOD of 0.24 ng/mL and recovery rates from 79 to 123%. Furthermore, we demonstrated the versatility and robustness of the assay by adapting it to the detection of two other clinically relevant biomarkers, prostate-specific antigen (PSA) and cardiac troponin (cTn), reaching the LODs in spiked serum of 9.4 pg/mL and 0.62 ng/mL for PSA and cTn, respectively. Finally, clinical samples of patients examined for prostate cancer were analyzed, achieving a strong correlation with the reference electrochemiluminescence immunoassay (recovery rates from 89 to 117%). The achieved results demonstrate that UCNPs are highly sensitive labels that enable the development of dot-blot immunoassays for quantitative analysis of low-abundance biomarkers.
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Biomarcadores , Límite de Detección , Nanopartículas , Antígeno Prostático Específico , Humanos , Inmunoensayo/métodos , Nanopartículas/química , Antígeno Prostático Específico/sangre , Antígeno Prostático Específico/análisis , Biomarcadores/sangre , Biomarcadores/orina , Biomarcadores/análisis , Puntos Cuánticos/química , Albúmina Sérica Humana/análisis , Albúmina Sérica Humana/orina , MasculinoRESUMEN
The three decades of experience with piezoelectric devices applied in the field of bioanalytical chemistry are shared. After introduction to principles and suitable measuring approaches, active and passive methods based on oscillators and impedance analysis, respectively, the focus is directed towards biosensing approaches. Immunosensing examples are provided, followed by other affinity sensing approaches based on hybridization of nucleic acids, aptamers, monitoring of enzyme activities, and detection of pathogenic microbes. The combination of piezosensors with cell lines and testing of drugs is highlighted, including mechanically active cells. The combination of piezosensors with other measuring techniques providing original hybrid devices is briefly discussed.
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Ácidos Nucleicos , Línea Celular , Impedancia Eléctrica , OligonucleótidosRESUMEN
Photon-upconversion nanoparticles (UCNP) have already been established as labels for affinity assays in analog and digital formats. Here, advanced, or smart, systems based on UCNPs coated with active shells, fluorescent dyes, and metal and semiconductor nanoparticles participating in energy transfer reactions are reviewed. In addition, switching elements can be embedded in such assemblies and provide temporal and spatial control of action, which is important for intracellular imaging and monitoring activities. Demonstration and critical comments on representative approaches demonstrating the progress in the use of such UCNPs in bioanalytical assays, imaging, and monitoring of target molecules in cells are reported, including particular examples in the field of cancer theranostics.
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Nanopartículas , Fotones , Humanos , Nanopartículas/química , Colorantes Fluorescentes/química , Animales , Neoplasias/diagnóstico por imagen , Neoplasias/diagnóstico , Imagen ÓpticaRESUMEN
The COVID-19 crisis requires fast and highly sensitive tests for the early stage detection of the SARS-CoV-2 virus. For detecting the nucleocapsid protein (N protein), the most abundant viral antigen, we have employed upconversion nanoparticles that emit short-wavelength light under near-infrared excitation (976 nm). The anti-Stokes emission avoids autofluorescence and light scattering and thus enables measurements without optical background interference. The sandwich upconversion-linked immunosorbent assay (ULISA) can be operated both in a conventional analog mode and in a digital mode based on counting individual immune complexes. We have investigated how different antibody combinations affect the detection of the wildtype N protein and the detection of SARS-CoV-2 (alpha variant) in lysed culture fluid via the N protein. The ULISA yielded a limit of detection (LOD) of 1.3 pg/mL (27 fM) for N protein detection independent of the analog or digital readout, which is approximately 3 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays or commercial lateral flow assays for home testing. In the case of SARS-CoV-2, the digital ULISA additionally improved the LOD by a factor of 10 compared to the analog readout.
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COVID-19 , Inmunoadsorbentes , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Nucleocápside , Anticuerpos Antivirales , Sensibilidad y EspecificidadRESUMEN
RATIONALE: Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. OBJECTIVES: We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms. METHODS AND RESULTS: We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFß1 (transforming growth factor ß1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. CONCLUSIONS: Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.
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Cardiomiopatía Dilatada/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Insuficiencia Cardíaca/metabolismo , Infarto del Miocardio/metabolismo , Miocardio/metabolismo , Función Ventricular Izquierda , Remodelación Ventricular , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Estudios de Casos y Controles , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Matriz Extracelular/genética , Matriz Extracelular/ultraestructura , Fibroblastos/ultraestructura , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Mecanotransducción Celular , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/ultraestructura , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Señalizadoras YAPRESUMEN
An electrochemical impedimetric biosensor for human serum albumin (HSA) determination is proposed. The biosensor is based on water-phase assembled nanocomposites made of 2D WS2 nanoflakes and Au nanoparticles (AuNPs). The WS2 has been produced using a liquid-phase exfoliation strategy assisted by sodium cholate, obtaining a water-stable suspension that allowed the straightforward decoration with AuNPs directly in the aqueous phase. The resulting WS2/Au nanocomposite has been characterized by atomic force microscopy and Raman spectroscopy and, then, employed to modify screen-printed electrodes. Good electron-transfer features have been achieved. An electrochemical immunosensing platform has been assembled exploiting cysteamine-glutaraldehyde covalent chemistry for antibody (Ab) immobilization. The resulting immunosensor exhibited good sensitivity for HSA detection (LOD = 2 ng mL-1), with extended linear range (0.005 - 100 µg mL-1), providing a useful analytical tool for HSA determination in urine at relevant clinical ranges for microalbuminuria screening. The HSA quantification in human urine samples resulted in recoveries from 91.8 to 112.4% and was also reproducible (RSD < 7.5%, n = 3), with marked selectivity. This nanocomposite, thanks to the reliable performance and the ease of the assembling strategy, is a promising alternative for electrochemical immunosensing of health relevant markers.
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Técnicas Biosensibles , Nanopartículas del Metal , Nanocompuestos , Humanos , Nanopartículas del Metal/química , Agua , Oro/química , Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Albúmina Sérica Humana , Nanocompuestos/químicaRESUMEN
Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labelsâhorseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)âUCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection.
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Inmunoadsorbentes , Nanopartículas , Masculino , Humanos , Inmunoensayo/métodos , Límite de Detección , Estreptavidina , MagnetismoRESUMEN
Immunohistochemistry (IHC) and immunocytochemistry (ICC) are widely used to identify cancerous cells within tissues and cell cultures. Even though the optical microscopy evaluation is considered the gold standard, the limited range of useful labels and narrow multiplexing capabilities create an imminent need for alternative readout techniques. Laser-induced breakdown spectroscopy (LIBS) enables large-scale multi-elemental analysis of the surface of biological samples, e.g., thin section or cell pellet. It is, therefore, a potential alternative for IHC and ICC readout of various labels or tags (Tag-LIBS approach). Here, we introduce Tag-LIBS as a method for the specific determination of HER2 biomarker. The cell pellets were labeled with streptavidin-conjugated upconversion nanoparticles (UCNP) through a primary anti-HER2 antibody and a biotinylated secondary antibody. The LIBS scanning enabled detecting the characteristic elemental signature of yttrium as a principal constituent of UCNP, thus indirectly providing a reliable way to differentiate between HER2-positive BT-474 cells and HER2-negative MDA-MB-231 cells. The comparison of results with upconversion optical microscopy and luminescence intensity scanning confirmed that LIBS is a promising alternative for the IHC and ICC readout.
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Biomarcadores de Tumor/análisis , Nanopartículas/química , Receptor ErbB-2/análisis , Anticuerpos Inmovilizados/inmunología , Biomarcadores de Tumor/inmunología , Línea Celular Tumoral , Estudios de Factibilidad , Fluoruros/química , Fluoruros/efectos de la radiación , Humanos , Inmunohistoquímica/métodos , Luz , Nanopartículas/efectos de la radiación , Receptor ErbB-2/inmunología , Análisis Espectral/métodos , Tulio/química , Tulio/efectos de la radiación , Itrio/química , Itrio/efectos de la radiaciónRESUMEN
Numerous protocols of cardiac differentiation have been established by essentially focusing on specific growth factors on human pluripotent stem cell (hPSC) differentiation efficiency. However, the optimal environmental factors to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Here, we hypothesized that low oxygen exposure enhances the molecular and functional maturity of the cardiomyocytes. We aimed at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hPSCs in 2D- and 3D-based protocols. hPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed. The mitochondrial localization and metabolic properties were evaluated. The intracellular Ca2+ handling and contractile properties were also monitored. The 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac spheroids containing hPSC-CMs in LOE further exhibited cardiac markers, hypertrophy, steadier SR Ca2+ release properties revealing a better SR Ca2+ handling, and enhanced contractile force. Preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac spheroids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.
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Diferenciación Celular , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Oxígeno/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Biomarcadores , Calcio/metabolismo , Técnicas de Cultivo de Célula , Expresión Génica , Humanos , Mitocondrias Cardíacas/metabolismo , Retículo Sarcoplasmático/metabolismo , Esferoides CelularesRESUMEN
Lanthanide-doped upconversion nanoparticles (UCNPs) display highly beneficial photophysical features for background-free bioimaging and bioanalysis; however, they are instable in high ionic strength buffers, have no functional groups, and are nonspecifically interacting. Here, we have prepared NIR-excitable UCNPs that are long-term colloidally stable in buffered media and possess functional groups. Heterobifunctional poly(ethylene glycol) (PEG) linkers bearing neridronate and alkyne or maleimide were attached to UCNPs via a ligand exchange. Streptavidin (SA)-conjugates were prepared by click reaction of UCNP@PEG-alkyne and SA-azide. Antihuman serum albumin pAbF antibody was modified with azide groups and conjugated to UCNP@PEG-alkyne via click reaction; alternatively, the antibody, after mild reduction of its disulfide bonds, was conjugated to UCNP@PEG-maleimide. We employed these nanoconjugates as labels for an upconversion-linked immunosorbent assay. SA-based labels achieved the lowest LOD of 0.17 ng/mL for the target albumin, which was superior compared to a fluorescence immunoassay (LOD 0.59 ng/mL) or an enzyme-linked immunoassay (LOD 0.56 ng/mL).
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Nanopartículas , PolietilenglicolesRESUMEN
The ability to detect low concentrations of analytes and in particular low-abundance biomarkers is of fundamental importance, e.g., for early-stage disease diagnosis. The prospect of reaching the ultimate limit of detection has driven the development of single-molecule bioaffinity assays. While many review articles have highlighted the potentials of single-molecule technologies for analytical and diagnostic applications, these technologies are not as widespread in real-world applications as one should expect. This Review provides a theoretical background on single-molecule-or better digital-assays to critically assess their potential compared to traditional analog assays. Selected examples from the literature include bioaffinity assays for the detection of biomolecules such as proteins, nucleic acids, and viruses. The structure of the Review highlights the versatility of optical single-molecule labeling techniques, including enzymatic amplification, molecular labels, and innovative nanomaterials.
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Imagen Individual de Molécula/métodos , Sitios de Unión , Biomarcadores/análisis , Ensayo de Inmunoadsorción Enzimática , Colorantes Fluorescentes/química , Límite de Detección , Nanoestructuras/química , Ácidos Nucleicos/análisis , Reacción en Cadena de la Polimerasa/métodos , Proteínas/análisis , Relación Señal-Ruido , Virus/aislamiento & purificaciónRESUMEN
Single-molecule (digital) immunoassays provide the ability to detect much lower protein concentrations than conventional immunoassays. As photon-upconversion nanoparticles (UCNPs) can be detected without optical background interference, they are excellent labels for so-called single-molecule upconversion-linked immunosorbent assays (ULISAs). We have introduced a UCNP label design based on streptavidin-PEG-neridronate and a two-step detection scheme involving a biotinylated antibody that efficiently reduces nonspecific binding on microtiter plates. In a microtiter plate immunoassay, individual sandwich immune complexes of the cancer marker prostate-specific antigen (PSA) are detected and counted by wide-field epiluminescence microscopy (digital readout). The digital detection is 16× more sensitive than the respective analogue readout and thus expands the limit of detection to the sub-femtomolar concentration range (LOD: 23 fg mL-1, 800 aM). The single molecule ULISA shows excellent correlation with an electrochemiluminescence reference method. Although the analogue readout can routinely measure PSA concentrations in human serum samples, very low concentrations have to be monitored after radical prostatectomy. Combining the digital and analogue readout covers a dynamic range of more than 3 orders of magnitude in a single experiment.
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Inmunoensayo/métodos , Técnicas de Inmunoadsorción , Antígeno Prostático Específico/sangre , Imagen Individual de Molécula/métodos , Dermoscopía/métodos , Difosfonatos , Humanos , Masculino , Nanopartículas/química , Fotones , Polietilenglicoles , EstreptavidinaRESUMEN
Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca2+ handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca2+ handling, employing in vitro models of cardiomyocytes, has become a cornerstone to understand the role and adaptation of calcium signalling in healthy and diseased hearts. Comprehensive in vitro systems and cell-based biosensors are powerful tools to enrich and speed up cardiac phenotypic and drug response evaluation. We have implemented a combined setup to measure contractility and calcium waves in human embryonic stem cells-derived cardiomyocyte 3D clusters, obtained from embryoid body differentiation. A combination of atomic force microscopy to monitor cardiac contractility, and sensitive fast scientific complementary metal-oxide-semiconductor camera for epifluorescence video recording, provided correlated signals in real time. To speed up the integrated data processing, we tested several post-processing algorithms, to improve the automatic detection of relevant functional parameters. The validation of our proposed method was assessed by caffeine stimulation (10mM) and detection/characterization of the induced cardiac response. We successfully report the first simultaneous recording of cardiac contractility and calcium waves on the described cardiac 3D models. The drug stimulation confirmed the automatic detection capabilities of the used algorithms, measuring expected physiological response, such as elongation of contraction time and Ca2+ cytosolic persistence, increased calcium basal fluorescence, and transient peaks. These results contribute to the implementation of novel, integrated, high-information, and reliable experimental systems for cardiac models and drug evaluation.
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Biofisica/métodos , Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Señalización del Calcio/fisiología , HumanosRESUMEN
We review the progress achieved during the recent five years in immunochemical biosensors (immunosensors) combined with nanoparticles for enhanced sensitivity. The initial part introduces antibodies as classic recognition elements. The optical sensing part describes fluorescent, luminescent, and surface plasmon resonance systems. Amperometry, voltammetry, and impedance spectroscopy represent electrochemical transducer methods; electrochemiluminescence with photoelectric conversion constitutes a widely utilized combined method. The transducing options function together with suitable nanoparticles: metallic and metal oxides, including magnetic ones, carbon-based nanotubes, graphene variants, luminescent carbon dots, nanocrystals as quantum dots, and photon up-converting particles. These sources merged together provide extreme variability of existing nanoimmunosensing options. Finally, applications in clinical analysis (markers, tumor cells, and pharmaceuticals) and in the detection of pathogenic microorganisms, toxic agents, and pesticides in the environmental field and food products are summarized.
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Técnicas Biosensibles , Inmunoensayo , Nanopartículas/química , Animales , HumanosRESUMEN
We report on the successful application of carboxyl-rich plasma polymerized (PP) films as a matrix layer for bioreceptor immobilization in surface plasmon resonance (SPR) immunosensing. Composition and chemical properties of the carboxyl-rich PP films deposited from a mixture of maleic anhydride and acetylene were investigated. Changes in the films stored in air, water, and buffer were studied and the involved chemical changes were described. Performance in SPR immunosensing was evaluated on interactions of human serum albumin (HSA) with a specific monoclonal antibody. The comparison with the mixed self-assembled monolayer of mercaptoundecanoic acid and mercaptohexanol (MUA/MCH) and one of the most widely used surfaces for SPR, the 2D and 3D carboxymethylated dextran (CMD), was presented to show the efficacy of plasma polymerized matrix layers for biosensing. The PP film-based SPR immunosensor provided a similar detection limit of HSA (100 ng/mL) as MUA/MCH- (100 ng/mL) and 3D CMD (50 ng/mL)-based sensors. However, the response levels were about twice higher in case of the PP film-based immunosensor than in case of MUA/MCH-based alternative. The PP film surfaces had similar binding capacity towards antibody as the 3D CMD layers. The response of PP film-based sensor towards HSA was comparable to 3D CMD-based sensor up to 2.5 µg/mL. For the higher concentrations (> 10 µg/mL), the response of PP film-based immunosensor was lower due to inaccessibility of active sites of the immobilized antibody inside the flat PP film surface. We have demonstrated that due to its high stability and cost-effective straightforward preparation, the carboxyl-rich PP films represent an efficient alternative to self-assembled monolayers (SAM) and dextran-based layers in label-free immunosensing. Graphical abstract.
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Acetileno/química , Anhídridos Maleicos/química , Gases em Plasma , Polímeros/química , Resonancia por Plasmón de Superficie/métodos , Técnicas Biosensibles , Límite de Detección , Microscopía de Fuerza Atómica , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
This review (with 129 refs) summarizes the progress in electrochemical immunoassays combined with magnetic particles that was made in the past 5 years. The specifity of antibodies linked to electrochemical transduction (by amperometry, voltammetry, impedimetry or electrochemiluminescence) gains further attractive features by introducing magnetic nanoparticles (MNPs). This enables fairly easy preconcentration of analytes, minimizes matrix effects, and introduces an appropriate label. Following an introduction into the fundamentals of electrochemical immunoassays and on nanomaterials for respective uses, a large chapter addresses method for magnetic capture and preconcentration of analytes. A next chapter discusses commonly used labels such as dots, enzymes, metal and metal oxide nanoparticles and combined clusters. The large field of hybrid nanomaterials for use in such immunoassays is discussed next, with a focus on MNPs composites with various kinds of graphene variants, polydopamine, noble metal nanoparticles or nanotubes. Typical applications address clinical markers (mainly blood and urine parameters), diagnosis of cancer (markers and cells), detection of pathogens (with subsections on viruses and bacteria), and environmental and food contaminants as toxic agents and pesticides. A concluding section summarizes the present status, current challenges, and highlights future trends. Graphical abstract Magnetic nanoparticles (MNP) with antibodies (Ab) capture and preconcentrate analyte from sample (a) and afterwards become magnetically (b) or immunospecifically (c) bound at an electrode. Signal either increases due to the presence of alabel (b) or decreases as the redox probe is blocked (c).
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Electroquímica/métodos , Inmunoensayo/métodos , Imanes/química , Nanopartículas/química , Animales , HumanosRESUMEN
Laser-induced breakdown spectroscopy (LIBS) was examined as a novel method for readout of microtiter plate immunoassays involving nanoparticles (NP). The so-called Tag-LIBS technique is a sensitive method for the detection of specific biomarkers. It was applied to the determination of NP labels using nanosecond ablation sampling. The NP labels were examined from the bottom of a standard 96-well microtiter plate. Thanks to the flexibility of LIBS instrumentation, both the plasma emission collection and the focusing optics arrangements can be collinearly arranged. The experiments showed that silver NPs and gold NPs can be readily quantified on the bottom of the microtiter plate. Utilizing this technique, a sandwich immunoassay for human serum albumin using streptavidin-coated AgNP labels was developed. The assay has a 10 ng·mL-1 detection limit which is comparable to the sensitivity of fluorometric readout. The main advantage of this LIBS technique is its wide scope in which it enables a detection of almost any type of NP labels, irrespective to any fluorescence or catalytic properties. Owing to the immediate signal response, the relatively simple instrumentation also enables assay automation. The LIBS capability of multi-elemental analyses makes it a promising and fast alternative to other readout techniques, in particular with respect to multiplexed detection of biomarkers. Graphical abstract Laser-induced breakdown spectroscopy (LIBS) is used as a novel readout method of nanoparticle-based immunoassays in microtiter plates. After formation of sandwich immunocomplex, the analyte concentration is quantified as the signal of Ag nanoparticle labels determined by LIBS.
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Inmunoensayo/métodos , Rayos Láser , Nanopartículas del Metal/química , Albúmina Sérica Humana/análisis , Biomarcadores/sangre , Oro/química , Humanos , Tamaño de la Partícula , Plata/química , Propiedades de SuperficieRESUMEN
Enzyme immunoassays are widely used for detection of analytes within various samples. However, enzymes as labels suffer several disadvantages such as high production cost and limited stability. Catalytic nanoparticles (nanozymes) can be used as an alternative label in immunoassays overcoming the inherent disadvantages of enzymes. Prussian blue nanoparticles (PBNPs) are nanozymes composed of the Fe4[Fe(CN)6]3-based coordination polymer. They reveal peroxidase-like activity and are capable of catalyzing the oxidation of colorless 3,3',5,5'-tetramethylbenzidine in the presence of H2O2 to form intensely blue product. Here, we introduce the method for conjugation of PBNPs with antibodies and their application in nanozyme-linked immunosorbent assay (NLISA). Sandwich NLISA for detection of human serum albumin in urine was developed with limit of detection (LOD) of 1.2 ng·mL-1 and working range up to 1 µg·mL-1. Furthermore, the microbial contamination of Salmonella Typhimurium in powdered milk was detected with LOD of 6 × 103 colony-forming units (cfu)·mL-1 and working range up to 106 cfu·mL-1. In both cases, a critical comparison with the same immunoassay but using native peroxidase as label was realized. The achieved results confirmed the suitability of PBNPs for universal and robust replacement of enzyme labels.
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Técnicas Biosensibles/métodos , Ferrocianuros/química , Técnicas de Inmunoadsorción , Nanopartículas/química , Animales , Anticuerpos Antibacterianos/inmunología , Catálisis , Humanos , Límite de Detección , Leche/microbiología , Salmonella typhimurium/inmunología , Salmonella typhimurium/aislamiento & purificación , Albúmina Sérica Humana/inmunología , Albúmina Sérica Humana/orinaRESUMEN
The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL-1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.
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Inmunoensayo/métodos , Inmunoadsorbentes/química , Límite de Detección , Antígeno Prostático Específico/análisis , Biomarcadores/análisis , Inmunoadsorbentes/inmunología , Luminiscencia , Nanopartículas/químicaRESUMEN
Stem cell-derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient-specific human pluripotent stem cell (hPSC)-derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation-contraction coupling, which determines important functional properties of the hPSC-derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC-derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation-contraction coupling dynamics of the hPSC-derived CMs.