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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.
Assuntos
Biomarcadores , Limite de Detecção , Nanopartículas , Antígeno Prostático Específico , Humanos , Imunoensaio/métodos , Nanopartículas/química , Antígeno Prostático Específico/sangue , Antígeno Prostático Específico/análise , Biomarcadores/sangue , Biomarcadores/urina , Biomarcadores/análise , Pontos Quânticos/química , Albumina Sérica Humana/análise , Albumina Sérica Humana/urina , MasculinoRESUMO
Hyaluronic acid is an excellent biocompatible material for in vivo applications. Its ability to bind CD44, a cell receptor involved in numerous biological processes, predetermines HA-based nanomaterials as unique carrier for therapeutic and theranostic applications. Although numerous methods for the synthesis of hyaluronic acid nanoparticles (HANPs) are available today, their low reproducibility and wide size distribution hinder the precise assessment of the effect on the organism. A robust and reproducible approach for producing HANPs that meet strict criteria for in vivo applications (e.g., to lung parenchyma) remains challenging. We designed and evaluated four protocols for the preparation of HANPs with those required parameters. The HA molecule was cross-linked by novel combinations of carbodiimide, and four different amine-containing compounds resulted in monodisperse HANPs with a low polydispersity index. By a complex postsynthetic characterization, we confirmed that the prepared HANPs meet the criteria for inhaled therapeutic delivery and other in vivo applications.
Assuntos
Ácido Hialurônico , Nanopartículas , Ácido Hialurônico/química , Nanopartículas/química , Humanos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/síntese química , Animais , Tamanho da Partícula , Receptores de Hialuronatos/metabolismoRESUMO
Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor-acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).
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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.
Assuntos
Nanopartículas , Fótons , Humanos , Nanopartículas/química , Corantes Fluorescentes/química , Animais , Neoplasias/diagnóstico por imagem , Neoplasias/diagnóstico , Imagem ÓpticaRESUMO
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.
Assuntos
COVID-19 , Imunoadsorventes , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Ensaio de Imunoadsorção Enzimática , Proteínas do Nucleocapsídeo , Anticorpos Antivirais , Sensibilidade e EspecificidadeRESUMO
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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Imunoadsorventes , Nanopartículas , Masculino , Humanos , Imunoensaio/métodos , Limite de Detecção , Estreptavidina , MagnetismoRESUMO
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 Tumorais/análise , Nanopartículas/química , Receptor ErbB-2/análise , Anticorpos Imobilizados/imunologia , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Estudos de Viabilidade , Fluoretos/química , Fluoretos/efeitos da radiação , Humanos , Imuno-Histoquímica/métodos , Luz , Nanopartículas/efeitos da radiação , Receptor ErbB-2/imunologia , Análise Espectral/métodos , Túlio/química , Túlio/efeitos da radiação , Ítrio/química , Ítrio/efeitos da radiaçãoRESUMO
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 , PolietilenoglicóisRESUMO
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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Imagem Individual de Molécula/métodos , Sítios de Ligação , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática , Corantes Fluorescentes/química , Limite de Detecção , Nanoestruturas/química , Ácidos Nucleicos/análise , Reação em Cadeia da Polimerase/métodos , Proteínas/análise , Razão Sinal-Ruído , Vírus/isolamento & purificaçãoRESUMO
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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Imunoensaio/métodos , Técnicas de Imunoadsorção , Antígeno Prostático Específico/sangue , Imagem Individual de Molécula/métodos , Dermoscopia/métodos , Difosfonatos , Humanos , Masculino , Nanopartículas/química , Fótons , Polietilenoglicóis , EstreptavidinaRESUMO
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 Biossensoriais , Imunoensaio , Nanopartículas/química , Animais , HumanosRESUMO
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 , Anidridos Maleicos/química , Gases em Plasma , Polímeros/química , Ressonância de Plasmônio de Superfície/métodos , Técnicas Biossensoriais , Limite de Detecção , Microscopia de Força Atômica , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
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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Eletroquímica/métodos , Imunoensaio/métodos , Imãs/química , Nanopartículas/química , Animais , HumanosRESUMO
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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Imunoensaio/métodos , Lasers , Nanopartículas Metálicas/química , Albumina Sérica Humana/análise , Biomarcadores/sangue , Ouro/química , Humanos , Tamanho da Partícula , Prata/química , Propriedades de SuperfícieRESUMO
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 Biossensoriais/métodos , Ferrocianetos/química , Técnicas de Imunoadsorção , Nanopartículas/química , Animais , Anticorpos Antibacterianos/imunologia , Catálise , Humanos , Limite de Detecção , Leite/microbiologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/isolamento & purificação , Albumina Sérica Humana/imunologia , Albumina Sérica Humana/urinaRESUMO
We report on the toxicity and bioaccumulation of three different types of Cd-based quantum dots (QDs), dispersed in aqueous medium, for a model plant Allium cepa L. It is believed that encapsulation of nanoparticles should reduce their toxicity and increase their stability in different environments; in this work we studied how QD encapsulation affects their phytotoxicity. Core, core/shell, and core/shell/shell QDs (CdTe, CdTe/ZnS, and CdTe/CdS/ZnS QDs capped by 2-mercaptopropionic acid) were tested and CdCl2 was used as a positive control. After 24-h and 72-h exposure, total Cd content (MCd) and bioaccumulation factors (BAFs) were determined in all parts of A. cepa plants (roots, bulb, shoot), and the total length of the root system was monitored as a toxicity end-point. Measurements of total Cd content versus free Cd2+ content (with Differential Pulse Voltammetry, DPV) in exposure media showed differences in chemical stability of the three QD types. Correspondingly, selected QDs showed different toxicity for A. cepa and different Cd bioaccumulation patterns. CdTe QDs were the most toxic; their effect was similar to CdCl2 due to the release of free Cd2+, which was confirmed by the DPV measurements. Plants exposed to CdTe QDs also bioaccumulated the most Cd among all QD exposure groups. CdTe/ZnS QDs showed no toxicity and very low bioaccumulation of Cd in A. cepa; the main source of measured Cd in the plants were QDs adsorbed on their roots, which was confirmed by fluorescence microscopy. On the contrary, CdTe/CdS/ZnS QD toxicity and bioaccumulation patterns were similar to those of CdTe QDs and pointed to unstable CdS/ZnS shells.
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Compostos de Cádmio/análise , Compostos de Cádmio/toxicidade , Cebolas/efeitos dos fármacos , Pontos Quânticos/análise , Pontos Quânticos/toxicidade , Adsorção , Microscopia de Fluorescência , Cebolas/química , Raízes de Plantas/química , Raízes de Plantas/efeitos dos fármacosRESUMO
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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Imunoensaio/métodos , Imunoadsorventes/química , Limite de Detecção , Antígeno Prostático Específico/análise , Biomarcadores/análise , Imunoadsorventes/imunologia , Luminescência , Nanopartículas/químicaRESUMO
Contamination of food by pathogenic bacteria has always been a serious threat for human health. The amount of food exports and imports has been increasing in recent years which requires precise food quality control with short analysis time and simplified sample treatment. Surface plasmon resonance (SPR) immunosensor enhanced by biocatalyzed precipitation was developed for the analysis of Salmonella in dairy products. The specific capture antibody was immobilized on the SPR chip which allowed a direct label-free detection of Salmonella Typhimurium with the limit of detection (LOD) of 104 CFU·mL-1 and the analysis time of 10 min. Alternatively, the secondary detection antibody was conjugated with horseradish peroxidase to provide a signal enhancement by the biocatalyzed conversion of 4-chloro-1-naphthol to insoluble benzo-4-chlorocyclohexadienone. The formation of precipitate was studied in detail by atomic force microscopy (AFM). The sensitivity was increased 40 times in case of the precipitation-enhanced detection compared to the label-free approach. The optimized method provided LOD of 100 CFU·mL-1 with linear range up to 106 CFU·mL-1. The total time of analysis including bacteria binding and enhancement step was below 60 min. The capability to analyze real samples with complex matrices was demonstrated on the detection of Salmonella in powdered milk. The developed sensor represents simple and robust approach for routine monitoring of food contamination.
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Contaminação de Alimentos/análise , Imunoensaio , Leite/microbiologia , Salmonella typhimurium/isolamento & purificação , Ressonância de Plasmônio de Superfície , Animais , Microscopia de Força Atômica , Pós/químicaRESUMO
Photon-upconverting nanoparticles (UCNPs) emit light of shorter wavelength under near-infrared excitation and thus avoid optical background interference. We have exploited this unique photophysical feature to establish a sensitive competitive immunoassay for the detection of the pharmaceutical micropollutant diclofenac (DCF) in water. The so-called upconversion-linked immunosorbent assay (ULISA) was critically dependent on the design of the upconversion luminescent detection label. Silica-coated UCNPs (50 nm in diameter) exposing carboxyl groups on the surface were conjugated to a secondary anti-IgG antibody. We investigated the structure and monodispersity of the nanoconjugates in detail. Using a highly affine anti-DCF primary antibody, the optimized ULISA reached a detection limit of 0.05 ng DCF per mL. This performance came close to a conventional enzyme-linked immunosorbent assay (ELISA) without the need for an enzyme-mediated signal amplification step. The ULISA was further employed for analyzing drinking and surface water samples. The results were consistent with a conventional ELISA as well as liquid chromatography-mass spectrometry (LC-MS).
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Diclofenaco/análise , Imunoensaio/métodos , Imunoadsorventes/química , Poluentes Químicos da Água/análise , Água Potável/química , Nanopartículas/química , Tamanho da Partícula , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
Airborne microorganisms are a major cause of respiratory diseases. Detection of pathogenic bacteria in the form of bioaerosols is required not only in peacetime but also in the threat of biological attacks. The label-free and direct detection of aerosolized biological agents is presented here. A desktop bioaerosol chamber for safe work with aerosolized microbial cells was constructed, and its functionality was tested. The model organisms (Escherichia coli) were disseminated using an aerosol generator in the chamber filled with either common laboratory indoor air or sterile air. The particles from the generated aerosol were collected using the cyclone SASS 2300, suspended in buffer and then analyzed using the piezoelectric immunosensor modified with specific capture antibodies. The frequency shifts indicated presence of the model biological agent with limit of detection of 1.45 × 10(4) CFU·L(-1) of air. The total time from sample collection to detection was 16 min. The system was fully automated and controlled remotely through a local network.