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BACKGROUND: Long cardiac troponin T (cTnT) has been proposed to be a promising and more specific biomarker of acute myocardial infarction (AMI). As it represents a subfraction of circulating cTnT, detection of very low concentrations is a requirement. The aim of this study was to develop a novel, highly sensitive immunoassay for long cTnT. METHODS: A two-step sandwich-type immunoassay for long cTnT was developed, utilizing upconverting nanoparticles (UCNPs) as reporters. The limits of detection and quantitation were determined for the assay. Linearity and matrix effects were evaluated. Performance with clinical samples was assessed with samples from patients with non-ST elevation myocardial infarction (NSTEMI, n = 30) and end-stage renal disease (ESRD, n = 37) and compared to a previously developed time-resolved fluorescence (TRF)-based long cTnT assay and a commercial high-sensitivity cTnT assay. RESULTS: The novel assay reached a 28-fold lower limit of detection (0.40â ng/L) and 14-fold lower limit of quantitation (1.79â ng/L) than the previously developed TRF long cTnT assay. Li-heparin and EDTA plasma, but not serum, were found to be suitable sample matrixes for the assay. In a receiver operating characteristics curve analysis, the troponin ratio (long/total cTnT) determined with the novel assay showed excellent discrimination between NSTEMI and ESRD with an area under the curve of 0.986 (95% CI, 0.967-1.000). CONCLUSIONS: By utilizing upconversion luminescence technology, we developed a highly sensitive long cTnT assay. This novel assay can be a valuable tool for investigating the full potential of long cTnT as a biomarker for AMI. ClinicalTrials.gov Registration Number: NCT04465591.
Assuntos
Troponina T , Troponina T/sangue , Humanos , Imunoensaio/métodos , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/sangue , Biomarcadores/sangue , Masculino , Feminino , Limite de Detecção , Falência Renal Crônica/sangue , Luminescência , Pessoa de Meia-Idade , Idoso , Nanopartículas/químicaRESUMO
Upconversion nanoparticles (UCNPs) have been frequently applied in Förster resonance energy transfer (FRET) bioanalysis. However, the understanding of how surface coatings, bioconjugation, and dye-surface distance influence FRET biosensing performance has not significantly advanced. Here, we investigated UCNP-to-dye FRET DNA-hybridization assays in H2O and D2O using â¼24 nm large NaYF4:Yb3+,Er3+ UCNPs coated with thin layers of silica (SiO2) or poly(acrylic acid) (PAA). FRET resulted in strong distance-dependent PL intensity changes. However, the PL decay times were not significantly altered because of continuous Yb3+-to-Er3+ energy migration during Er3+-to-dye FRET. Direct bioconjugation of DNA to the thin PAA coating combined with the closest possible dye-surface distance resulted in optimal FRET performance with minor influence from competitive quenching by H2O. The better comprehension of UCNP-to-dye FRET was successfully translated into a microRNA (miR-20a) FRET assay with a limit of detection of 100 fmol in a 80 µL sample volume.
Assuntos
Técnicas Biossensoriais , Nanopartículas , Ácidos Nucleicos , Transferência Ressonante de Energia de Fluorescência , Dióxido de Silício , Técnicas Biossensoriais/métodosRESUMO
Förster Resonance Energy Transfer (FRET) between single molecule donor (D) and acceptor (A) is well understood from a fundamental perspective and is widely applied in biology, biotechnology, medical diagnostics, and bio-imaging. Lanthanide doped upconverting nanoparticles (UCNPs) have demonstrated their suitability as alternative donor species. Nevertheless, while they solve most disadvantageous features of organic donor molecules, such as photo-bleaching, spectral cross-excitation, and emission bleed-through, the fundamental understanding and practical realizations of bioassays with UCNP donors remain challenging. Among others, the interaction between many donor ions (in donor UCNP) and many acceptors anchored on the NP surface and the upconversion itself within UCNPs, complicate the decay-based analysis of D-A interaction. In this work, the assessment of designed virtual core-shell NP (VNP) models leads to the new designs of UCNPs, such as @Er, Yb@Er, Yb@YbEr, which are experimentally evaluated as donor NPs and compared to the simulations. Moreover, the luminescence rise and decay kinetics in UCNP donors upon RET is discussed in newly proposed disparity measurements. The presented studies help to understand the role of energy-transfer and energy migration between lanthanide ion dopants and how the architecture of core-shell UCNPs affects their performance as FRET donors to organic acceptor dyes.
Assuntos
Elementos da Série dos Lantanídeos , Nanopartículas , Transferência Ressonante de Energia de Fluorescência/métodos , Íons , LuminescênciaRESUMO
Upconverting nanoparticles are attractive reporters for immunoassays, because their high specific activity and lack of autofluorescence background enable their detection at extremely low concentrations. However, the sensitivity achieved with heterogeneous sandwich immunoassays using nanoparticle reporters is generally limited by the nonspecific binding of nanoparticle antibody conjugates to solid supports. In this study, we characterized plasma components associated with elevated nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticles in heterogeneous two-step sandwich immunoassays. Plasma was consecutively fractionated using various chromatographic methods by selecting after each step the fractions producing the highest nonspecific binding of upconverting nanoparticle conjugates in an immunoassay for cardiac troponin I. Finally, the proteins in the fractions associated with highest amount of nonspecific binding were separated by gel electrophoresis and identified with mass spectrometry. The results indicated that complement component C1q was present in the fractions associated with the highest signal from nonspecific binding. The interference was not limited to only poly(acrylic acid)-coated nanoparticles or certain antibody combination, but occurred more generally. The interference was removed by increasing the ionic strength of the assay buffer in the sample incubation step or by adding a negatively charged blocker to bind on positively charged C1q, suggesting that the interaction is mostly electrostatic. Hence, we assume that the interference is likely to affect various negatively charged nanoparticles. The identification of complement component C1q as the major interfering protein allows for more rational design of countermeasures in future immunoassay development utilizing nanoparticle reporters.
Assuntos
Imunoconjugados , Nanopartículas , Resinas Acrílicas , Anticorpos , Complemento C1q , Imunoensaio/métodos , Nanopartículas/químicaRESUMO
We present frequency encoded upconversion (FE-UPCON) widefield microscopy, an imaging approach that allows for multiplexed signal recovery based on frequency encoding of selected upconverted lanthanide ion emission rather than separation based on energy or time. FE-UPCON allows for the separation of luminescence from spectrally and spatially overlapping trivalent lanthanide ions (Ln3+) in upconversion nanoparticles (UCNPs). Utilizing the numerous electronic energy levels of Ln3+, one can generate a frequency encoded signal by periodic coexcitation with a secondary light source (modulated at a chosen frequency) that, for a particular wavelength, enhances the luminescence of the Ln3+ of interest. We demonstrate that it is possible to selectively image spectrally overlapping UCNPs co-doped with Yb3+/Ho3+ or Yb3+/Er3+ by FE-UPCON in cells up to 10 frames per second on a conventional widefield microscope with the simple extension of an additional secondary light source and a chopper wheel for modulation. Additionally, we show that FE-UPCON does not compromise sensitivity and that single UCNP detection is obtainable. FE-UPCON adds a new dimension (frequency space) for multiplexed imaging with UCNPs.
RESUMO
The performance of photon-upconversion nanoparticles (UCNPs) as background-free luminescent labels in bioanalytical applications strongly depends on the preparation of well-defined and water-dispersible nanoconjugates. We have exploited the separation power of agarose-gel electrophoresis to purify milligram amounts of homogeneous UCNPs covered with carboxylated silica, biotin, or streptavidin with recovery rates of 30 to 50%. Clusters containing discrete numbers of UCNPs were isolated from the gel and reanalyzed by agarose-gel electrophoresis, single-nanoparticle-upconversion microscopy, and additional complementary methods. The purified nanoconjugates improved conventional (analogue) bioaffinity assays and provided highly monodisperse conjugates for assays that rely on counting individual UCNPs (digital assays).
Assuntos
Substâncias Luminescentes/isolamento & purificação , Nanopartículas Metálicas/química , Biotina/química , Eletroforese em Gel de Ágar/métodos , Európio/química , Raios Infravermelhos , Substâncias Luminescentes/química , Substâncias Luminescentes/efeitos da radiação , Nanopartículas Metálicas/efeitos da radiação , Tamanho da Partícula , Dióxido de Silício/química , Estreptavidina/químicaRESUMO
In this work, we explore a photochemical ligation reaction to covalently modify oligonucleotide-conjugated upconverting nanoparticles (UCNPs) in the presence of a specific target DNA sequence. The target sequence acts as a hybridization template, bringing together a biotinylated photoactivatable oligonucleotide probe and the oligonucleotide probe that is attached to UCNPs. The illumination of the UCNPs by NIR light to generate UV emission internally or illuminating the photoactivatable probe directly by an external UV light promotes the photochemical ligation reaction, yielding covalently biotin functionalized UCNPs that can be selectively captured in streptavidin-coated microwells. Following this strategy, we developed a DNA sensor with a limit of detection of 1 × 10-18 mol per well (20 fM). In addition, we demonstrate the possibility to create UCNP patterns on the surface of solid supports upon NIR illumination that are selectively formed under the presence of the target oligonucleotide.
Assuntos
Sondas de DNA/química , DNA/análise , Fluoretos/química , Nanopartículas/química , Ítrio/química , Resinas Acrílicas/química , Técnicas Biossensoriais/métodos , DNA/genética , Sondas de DNA/genética , Fluoretos/efeitos da radiação , Raios Infravermelhos , Nanopartículas/efeitos da radiação , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Túlio/química , Túlio/efeitos da radiação , Raios Ultravioleta , Itérbio/química , Itérbio/efeitos da radiação , Ítrio/efeitos da radiaçãoRESUMO
Fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) have enabled biologists to study processes of transport, binding, and enzymatic reactions in living cells. However, applying FCS and FCCS to samples such as whole blood and plasma is complicated as the fluorescence bursts of diffusing labels can be swamped by strong autofluorescence. Here we present cross-correlation spectroscopy based on two upconversion nanoparticles emitting at different wavelengths on the anti-Stokes side of a single excitation laser. This upconversion cross-correlation spectroscopy (UCCS) approach allows us to completely remove all Stokes shifted autofluorescence background in biological material such as plasma. As a proof of concept, we evaluate the applicability of UCCS to a homogeneous sandwich immunoassay for thyroid stimulating hormone measured in buffer solution and in plasma.
Assuntos
Corantes Fluorescentes/química , Imunoensaio/métodos , Metais Terras Raras/química , Nanopartículas/química , Espectrometria de Fluorescência/métodos , Difusão , Humanos , Lasers , Tamanho da Partícula , Fótons , Propriedades de Superfície , Tireotropina/sangueRESUMO
Aqueous solutions are the basis for most biomedical assays, but they quench the upconversion luminescence significantly. Surface modifications of upconverting nanoparticles are vital for shielding the obtained luminescence. Modifications also provide new possibilities for further use by introducing attaching sites for biomolecule conjugation. We demonstrate the use of a layer-by-layer surface modification method combining varying lengths of negatively charged polyelectrolytes with positive neodymium ions in coating the upconverting NaYF4:Yb3+,Er3+ nanoparticles. We confirmed the formation of the bilayers and investigated the surface properties with Fourier transform infrared and reflectance spectroscopy, thermal analysis, and ζ-potential measurements. The effect of the coating on the upconversion luminescence properties was characterized, and the bilayers with the highest improvement in emission intensity were identified. In addition, studies for the nanoparticle and surface stability were carried out in aqueous environments. It was observed that the bilayers were able to shield the materials' luminescence from quenching also in the presence of phosphate buffer that is currently considered the most disruptive environment for the nanoparticles.
RESUMO
Upconverting nanoparticles (UCNPs) are attractive reporters in immunoassays because of their outstanding detectability. However, non-specific binding of antibody-UCNP conjugates on protein coated solid support results in background, which limits the immunoassay sensitivity. Thus, the full potential of UCNPs as reporters cannot be fully exploited. The authors report here a method to improve the sensitivity of UCNP-based immunoassays by reducing the non-specific binding of antibody-UNCP conjugates on the protein coated solid support. In the assays studied here, poly(acrylic acid) (PAA) coated NaYF4:Yb3+,Er3+ type UCNPs were conjugated to two different antibodies against cardiac troponin I (cTnI) and thyroid stimulating hormone (TSH). The two-step heterogeneous sandwich immunoassays were performed in microtitration wells, and the green luminescence of antibody-UCNP conjugates was measured at 540 nm upon 980 nm excitation. Non-specific binding of antibody-UCNP conjugates was reduced by mixing free PAA with PAA coated UCNPs before adding the UCNPs to the wells. The free PAA in the buffer reduced the background in both cTnI and TSH immunoassays (compared to the control assay without free PAA). The limits of detection decreased from 2.1 ng·L-1 to 0.48 ng·L-1 in case of cTnI and from 0.070 mIU·L-1 to 0.020 mIU·L-1 in case of TSH if PAA is added to the buffer. Presumably, the effect of free PAA is due to blocking of the surface areas where PAA coated UCNP would bind proteins non-specifically. The method introduced here is likely to be applicable to other kinds of PAA-coated nanoparticles, and similar approaches conceivably work also with other nanoparticle coatings. Graphical abstract The presence of free poly(acrylic acid) (PAA) in a buffer solution prevents aggregation and non-specific protein binding of PAA-coated upconverting nanoparticles (UCNPs) in heterogeneous sandwich immunoassays. The decrease in non-specific binding enables distinctly more sensitive assays to be performed.
Assuntos
Resinas Acrílicas/química , Imunoensaio/métodos , Limite de Detecção , Nanopartículas/química , CalibragemRESUMO
Long-lifetime luminescent Eu(III) complexes are widely used as donors in Förster resonance energy transfer to enable time-gated detection of sensitized emission from an intrinsically short-lived acceptor. Here we report a unique energy-transfer system, where the sensitized acceptor emission has prolonged luminescence lifetime compared to the donor and the long lifetime is not cut short upon high energy-transfer efficiency. The infrared-excited, ultraviolet-emitting, Tm(III)-doped upconverting nanoparticles were used as donors, and a luminescent Eu(III)-chelate was used as an acceptor. Upon excitation the sensitized acceptor emission, which is already spectrally resolved from the donor, can be measured even after the donor luminescence has decayed. Because of anti-Stokes characteristics, the time-gated detection is not needed to avoid the autofluorescence. Thus, the long luminescence lifetime can be further modulated and utilized, e.g., in background-free molecular sensing, rendering the system extremely attractive.
RESUMO
Lysinuric protein intolerance (LPI) is a rare autosomal recessive disorder affecting the transport of cationic amino acids. Elevated plasma zinc concentrations have been described in patients with LPI. Calprotectin is a calcium- and zinc-binding protein, produced by polymorphonuclear leukocytes and monocytes. Both zinc and calprotectin have an important role in immune system. In this study, we describe plasma zinc and plasma calprotectin concentrations in Finnish LPI patients. Plasma calprotectin concentration was measured from 10 LPI patients using an enzyme-linked immunosorbent assay (ELISA) and it was remarkably high in all LPI patients (median: 622 338 µg/L) compared to that in healthy controls (608 µg/L). Plasma zinc concentration was measured by photometry and it was normal or only mildly elevated (median: 14.9 µmol/L). All the patients had decreased glomerular infiltration rate (median: 50 mL/min/1.73 m2). In conclusion, we observed extremely high plasma calprotectin concentration in patients with LPI. Mechanism of this phenomenon is unknown.
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Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.
Assuntos
Técnicas Biossensoriais , Micotoxinas , Imunoensaio/métodos , Micotoxinas/análise , Peptídeos/química , Transferência de EnergiaRESUMO
A quantitative multianalyte immunoassay utilizing luminescent upconverting single-crystal nanoparticles as reporters on an antibody array-in-well platform was demonstrated. Upconverting nanoparticles are inorganic rare earth doped materials that have the unique feature of converting low energy infrared radiation into higher energy visible light. Autofluorescence, commonly limiting the sensitivity of fluorescence-based assays, can be completely eliminated with photon upconversion technology because the phenomenon does not occur in biological materials. Biotinylated antibodies for three analytes (prostate specific antigen, thyroid stimulating hormone, and luteinizing hormone) were printed in an array format onto the bottom of streptavidin-coated microtiter wells. Analyte dilutions were added to the wells, and the analytes were detected with antibody-coated upconverting nanoparticles. Binding of the upconverting nanoparticles was imaged with an anti-Stokes photoluminescence microwell imager, and the standard curves for each analyte were quantified from the selected spot areas of the images. Single analyte and reference assays were also carried out to compare with the results of the multianalyte assay. Multiplexing did not have an effect on the assay performance. This study demonstrates the feasibility of upconverting single-crystal nanoparticles for imaging-based detection of quantitative multianalyte assays.
Assuntos
Imunoensaio/instrumentação , Substâncias Luminescentes/química , Hormônio Luteinizante/análise , Nanopartículas/química , Antígeno Prostático Específico/análise , Tireotropina/análise , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Biotinilação , Feminino , Humanos , Medições Luminescentes/instrumentação , Hormônio Luteinizante/imunologia , Masculino , Antígeno Prostático Específico/imunologia , Análise Serial de Proteínas/instrumentação , Sensibilidade e Especificidade , Tireotropina/imunologiaRESUMO
Lanthanide-doped colloidal nanoparticles capable of photon upconversion (UC) offer long luminescence lifetimes, narrowband absorption and emission spectra, and efficient anti-Stokes emission. These features are highly advantageous for Förster Resonance Energy Transfer (FRET) based detection. Upconverting nanoparticles (UCNPs) as donors may solve the existing problems of molecular FRET systems, such as photobleaching and limitations in quantitative analysis, but these new labels also bring new challenges. Here we have studied the impact of the core-shell compositional architecture of upconverting nanoparticle donors and the mode of photoexcitation on the performance of UC-FRET from UCNPs to Rose Bengal (RB) molecular acceptor. We have quantitatively compared luminescence rise and decay kinetics of Er3+ emission using core-only NaYF4: 20% Yb, 2% Er and core-shell NaYF4: 20% Yb @ NaYF4: 20% Yb, 5% Er donor UCNPs under three photoexcitation schemes: (1) direct short-pulse photoexcitation of Er3+ at 520 nm; indirect photoexcitation of Er3+ through Yb3+ sensitizer with (2) 980 nm short (5-7 ns) or (3) 980 nm long (4 ms) laser pulses. The donor luminescence kinetics and steady-state emission spectra differed between the UCNP architectures and excitation schemes. Aiming for highly sensitive kinetic upconversion FRET-based biomolecular assays, the experimental results underline the complexity of the excitation and energy-migration mechanisms affecting the Er3+ donor responses and suggest ways to optimize the photoexcitation scheme and the architecture of the UCNPs used as luminescent donors.
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BACKGROUND AND AIMS: Upconverting nanoparticles (UCNPs) are attractive reporters for immunoassays due to their excellent detectability. Assays sensitive enough to measure baseline level of cardiac troponin I cTnI in healthy population could be used to identify patients at risk for cardiovascular disease. Aiming for a cTnI assay of such sensitivity, the surface chemistry of the nanoparticles as well as the assay reagents and the protocol were optimized for monodispersity of the UCNP antibody conjugates (Mab UCNPs) and to minimize their non-specific interactions with the solid support. MATERIALS AND METHODS: UCNPs were coated with poly(acrylic acid) via two-step ligand exchange and conjugated with monoclonal antibodies. The conjugates were applied in a microplate-based sandwich immunoassay using a combination of two capture antibodies to detect cTnI. Assay was evaluated according to guidelines of Clinical & Laboratory Standards Institute. RESULTS: The limit of detection and limit of blank of the assay were 0.13 ng/L and 0.01 ng/L cTnI, respectively. The recoveries were >90% in spiked plasma in the linear range. The within- and between-run imprecisions were <10%. CONCLUSION: The results demonstrate that UCNPs enable quantification of cTnI concentrations expected in plasma of healthy individuals and could be used to identify patients at risk for cardiovascular disease.
Assuntos
Nanopartículas , Troponina I , Humanos , Imunoensaio , Testes Imunológicos , FótonsRESUMO
In this study, a multiplex serological array-in-well assay was constructed for simultaneous detection of serum IgG antibodies against parvovirus B19 and human adenovirus. The array was prepared in streptavidin-coated 96-well microtiter plates by spotting biotinylated parvovirus B19 virus-like-particles, adenovirus type 2 and 5 hexon antigens, negative control of human serum albumin and positive controls of human IgG and anti-human IgG antibodies on the bottom of each well in an array format with a printable area of 2 mm × 2 mm. The array-in-well assay was evaluated with serum samples (n=89) of different antibody status as determined by commercial enzyme immunoassay for parvovirus IgG, and by in-house enzyme immunoassay for adenovirus IgG. The bound serum anti-parvovirus IgG, anti-adenovirus IgG, and total IgG antibodies were detected with anti-human IgG antibody coated photon upconverting nanoparticles and the assay was measured with an anti-Stokes photoluminescence imager. Detection of specific antibodies by the multiplex array-in-well assay was in good agreement (100% for parvovirus B19 and 96% for adenovirus) with the reference results. In conclusion, the array-in-well with upconverting phosphor reporter technology was able to detect antiviral antibodies in human sera, and represents an efficient serodiagnostic concept that is a promising new tool for multiplex serology.
Assuntos
Infecções por Adenovirus Humanos/diagnóstico , Adenovírus Humanos/imunologia , Anticorpos Antivirais/sangue , Imunoglobulina G/sangue , Infecções por Parvoviridae/diagnóstico , Parvovirus B19 Humano/imunologia , Testes Sorológicos/métodos , Adulto , Pré-Escolar , Humanos , Medições LuminescentesRESUMO
Homogeneous assays are advantageous because of their simplicity and rapid kinetics but typically their performance is severely compromised compared to heterogeneous assay formats. Here, we report a homogeneous immunoassay utilizing switchable lanthanide luminescence for detection and site-specifically labeled recombinant antibody fragments as binders to improve the assay performance. Switchable lanthanide luminescence enabled elimination of assay background due to division of the luminescent lanthanide chelate into two non-luminescent label moieties. Simultaneous biomolecular recognition of model analyte cardiac troponin I by two antibody fragments brought the label moieties together and resulted in self-assembly of luminescent mixed chelate complex. The assay was very rapid as maximal signal-to-background ratios were achieved already after 6 min of incubation. Additionally, the limit of detection was 0.38 ng/mL (16 pM), which was comparable to the limit of detection for the heterogeneous reference assay based on the same binders (0.26 ng/mL or 11 pM). This is the first study to apply switchable lanthanide luminescence in immunoassays and demonstrates the versatile potential of the technology for rapid and sensitive homogeneous assays.