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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.
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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
Multiplexed lateral flow assays (LFAs) offer efficient on-site testing by simultaneously detecting multiple biomarkers from a single sample, reducing costs. In cancer diagnostics, where biomarkers can lack specificity, multiparameter detection provides more information at the point-of-care. Our research focuses on epithelial ovarian cancer (EOC), where STn-glycosylated forms of CA125 and CA15-3 antigens can better discriminate cancer from benign conditions. We have developed a dual-label LFA that detects both CA125-STn and CA15-3-STn within a single anti-STn antibody test line. This utilizes spectral separation of green (540 nm) and blue (450 nm) emitting erbium (NaYF4:Yb3+, Er3+)- and thulium (NaYF4: Yb3+, Tm3+)-doped upconverting nanoparticle (UCNP) reporters conjugated with antibodies against the protein epitopes in CA125 or CA15-3. This technology allows the simultaneous detection of different antigen variants from a single test line. The developed proof-of-concept dual-label LFA was able to distinguish between the ascites fluid samples from diagnosed ovarian cancer patients (n = 10) and liver cirrhosis ascites fluid samples (n = 3) used as a negative control. The analytical sensitivity of CA125-STn for the dual-label LFA was 1.8 U/ml in buffer and 3.6 U/ml in ascites fluid matrix. Here we demonstrate a novel approach of spectrally separated measurement of STn-glycosylated forms of two different cancer-associated protein biomarkers by using UCNP reporter technology.
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Antígeno Ca-125 , Proteínas de Membrana , Mucina-1 , Neoplasias Ovarianas , Humanos , Antígeno Ca-125/análise , Feminino , Neoplasias Ovarianas/diagnóstico , Glicosilação , Biomarcadores Tumorais/análise , Antígenos Glicosídicos Associados a Tumores/análise , Medições Luminescentes/métodos , Carcinoma Epitelial do Ovário/diagnóstico , Imunoensaio/métodosRESUMO
The development of sensitive point-of-care (POC) assay platforms is of interest for reducing the cost and time of diagnostics. Lateral flow assays (LFAs) are the gold standard for POC systems, but their sensitivity as such is inadequate, for example, in the case of cardiac diagnostics. The performance can be improved by incorporating different steps, such as pre-incubation to prolong the interaction time between sample and reporter for immunocomplex formation, and washing steps for background reduction. However, for POC assays, manual steps by the assay conductor are not desired. In this research, upconverting nanoparticles (UCNPs) were coated with poly(acrylic acid) (PAA) and conjugated to anti-cTnI antibodies, yielding non-clustering particles with low non-specific binding. The performance of cTnI-LFA in the PAA-anti-cTnI-UCNPs was compared to the same UCNPs with a commercial carboxyl surface. A kitchen-timer mechanism was embedded in a 3D-printed housing to produce a low-cost actuator facilitating a timed pre-incubation step for reporter and sample, and a washing step, to enable a multi-step cTnI-LFA with minimized manual labour. PAA-UCNPs showed improved mobility on nitrocellulose compared to those with a commercial surface. The mechanical actuator system was shown to improve sensitivity compared to a labour-intensive multi-step dipstick method, despite pre-incubation occurring during shaking and heating in the dipstick method. The limit of detection decreased from 7.6 to 1.5 ng/L cTnI in human plasma. The presented actuator can be easily modified for sensitivity improvement in the LFA for different analytes via pre-incubation and washing steps.
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Nanopartículas , Humanos , Imunoensaio/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Troponina I , Automação , Impressão TridimensionalRESUMO
Advances in the development of new biorecognition elements, nanoparticle-based labels as well as instrumentation have inspired the design of new bioaffinity assays. This review critically discusses the potential of nanoparticles to replace current enzymatic or molecular labels in immunoassays and other bioaffinity assays. Successful implementations of nanoparticles in commercial assays and the need for rapid tests incorporating nanoparticles in different roles such as capture support, signal generation elements, and signal amplification systems are highlighted. The limited number of nanoparticles applied in current commercial assays can be explained by challenges associated with the analysis of real samples (e.g., blood, urine, or nasal swabs) that are difficult to resolve, particularly if the same performance can be achieved more easily by conventional labels. Lateral flow assays that are based on the visual detection of the red-colored line formed by colloidal gold are a notable exception, exemplified by SARS-CoV-2 rapid antigen tests that have moved from initial laboratory testing to widespread market adaption in less than two years.
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Nanopartículas , Imunoensaio , Sensibilidade e EspecificidadeRESUMO
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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Pediatric neck infections and their complications, such as abscesses extending to deep neck compartments, are potentially life-threatening acute conditions. Medical imaging aims to verify abscesses and their extensions and exclude other complications. Magnetic resonance imaging (MRI) has proven to be a useful and highly accurate imaging method in acute neck infections in children. Children and adults differ in terms of the types of acute infections and the anatomy and function of the neck. This pictorial review summarizes typical findings in pediatric patients with neck infections and discusses some difficulties related to image interpretation.
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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.
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Técnicas Biossensoriais , Micotoxinas , Imunoensaio/métodos , Micotoxinas/análise , Peptídeos/química , Transferência de EnergiaRESUMO
The frequency-domain (FD) method provides an alternative to the commonly used time-domain (TD) approach in characterizing the luminescence kinetics of luminophores, with its own strengths, e.g., the capability to decouple multiple lifetime components with higher reliability and accuracy. While extensively explored for characterizing luminophores with down-shifted emission, this method has not been investigated for studying nonlinear luminescent materials such as lanthanide-doped upconversion nanoparticles (UCNPs), featuring more complicated kinetics. In this work, employing a simplified rate-equation model representing a standard two-photon energy-transfer upconversion process, we thoroughly analyzed the response of the luminescence of UCNPs in the FD method. We found that the FD method can potentially obtain from a single experiment the effective decay rates of three critical energy states of the sensitizer/activator ions involved in the upconversion process. The validity of the FD method is demonstrated by experimental data, agreeing reasonably well with the results obtained by TD methods.
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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.
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Técnicas Biossensoriais , Nanopartículas , Ácidos Nucleicos , Transferência Ressonante de Energia de Fluorescência , Dióxido de Silício , Técnicas Biossensoriais/métodosRESUMO
Infection of the deep neck spaces is a life-threatening acute illness that requires prompt diagnosis and treatment. Magnetic resonance imaging (MRI) offers unsurpassed soft tissue discrimination and is therefore well suited for imaging neck infections. Recently, the feasibility, diagnostic accuracy, and clinical significance of this method have been documented in patients with acute neck infections. This review article summarizes the scientific evidence, provides a practical guide to image acquisition and interpretation, reviews the most common imaging findings, and discusses some difficult diagnoses and pitfalls in acute neck infections, to help both radiologists and clinicians in managing these critically ill patients.
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OBJECTIVES: To determine the diagnostic accuracy of emergency magnetic resonance imaging (MRI) in odontogenic maxillofacial infections, the clinical and surgical significance of MRI findings, and whether MRI can identify the tooth responsible for the infection. METHODS: A retrospective cohort study reviewed 106 emergency neck MRI scans of patients with neck infections of odontogenic origin. The diagnostic accuracy of MRI in identifying abscesses was studied relative to surgical findings. Correlations were analyzed between various MRI findings and clinical results and outcomes, such as the surgical approach (intraoral vs. extraoral). The ability of MRI findings to predict the causative tooth was assessed in a blinded multi-reader setting. RESULTS: Of the 106 patients with odontogenic infections, 77 (73%) had one or more abscesses. Imaging showed a sensitivity, specificity, and accuracy of 0.95, 0.84, and 0.92, respectively, for MRI diagnosis of an odontogenic abscess. Among the imaging findings, mediastinal edema was the strongest predictor of extraoral surgery. MRI showed bone marrow edema in the majority of patients, and multi-reader assessment showed good reliability. MRI was also able to predict the causative tooth accurately. CONCLUSIONS: Emergency neck MRI can accurately detect odontogenic abscesses and reliably point to the causative tooth. These results can increase the utility and reliance on emergency MRI in clinical decision-making.
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Abscesso , Imageamento por Ressonância Magnética , Humanos , Abscesso/etiologia , Estudos Retrospectivos , Reprodutibilidade dos Testes , Imageamento por Ressonância Magnética/efeitos adversos , Edema/complicaçõesRESUMO
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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Immunoassays are important tools for clinical diagnosis as well as environmental and food analysis because they enable highly sensitive and quantitative measurements of analyte concentrations. In the 1980s, Roger Ekins suggested to improve the sensitivity of immunoassays by employing microspot assays, which are carried out under ambient analyte conditions and do not change the bulk analyte concentration of a sample during a measurement. More recently, the measurement of single analyte molecules has additionally attracted wide research interest. Although the ability to detect a single analyte molecule is not synonymous with the highest analytical sensitivity, single-molecule detection makes new routes accessible to avoiding background noise. This perspective follows the development of solid-phase immunoassays from the design of label techniques to single-molecule (digital) assays against the backdrop of Ekins's fundamental work on immunoassay theory. The essential aspects of both ambient analyte and digital assay approaches are presented as a guideline to finding a balance between the speed, sensitivity, and precision of immunoassays.
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Imunoensaio , Imunoensaio/métodosRESUMO
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.
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Imunoconjugados , Nanopartículas , Resinas Acrílicas , Anticorpos , Complemento C1q , Imunoensaio/métodos , Nanopartículas/químicaRESUMO
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.
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Elementos da Série dos Lantanídeos , Nanopartículas , Transferência Ressonante de Energia de Fluorescência/métodos , Íons , LuminescênciaRESUMO
BACKGROUND: Differences in the functioning of the immune system and the anatomical proportions of the neck between children and adults lead to different manifestations of deep neck infections. Magnetic resonance imaging (MRI) may serve as an alternative to computed tomography (CT) as the primary imaging modality. OBJECTIVE: To study characteristic MRI findings and the diagnostic accuracy of MRI in pediatric deep neck infections. MATERIALS AND METHODS: We retrospectively studied a cohort of pediatric patients who underwent a neck 3-tesla MRI study over a five-year period. Inclusion criteria were: 1) emergency MRI findings indicating an infection, 2) infection as the final clinical diagnosis, 3) diagnostic image quality verified by the radiologist reading the study and 4) age under 18 years. Patient record data, including surgery reports, were compared with the MRI findings. RESULTS: Data of 45 children were included and analysed. Compared to adults, children had a higher incidence of retropharyngeal infection and lymphadenitis, and a lower incidence of peritonsillar/parapharyngeal infection. MRI showed evidence of an abscess in 34 children. Of these 34 patients, 24 underwent surgery, which confirmed an abscess in 21 but no abscess in three patients. In addition, three patients underwent surgery without MRI evidence of abscess, and an abscess was found in one of these cases. The measures of diagnostic accuracy among the children were sensitivity 0.96, specificity 0.77, positive predictive value 0.89, negative predictive value 0.91 and accuracy 0.89. Compared with adults, children had lower C-reactive protein, but a similar proportion of them had an abscess, and abscess size and rate of surgery were similar. CONCLUSION: Despite the differences in the infection foci, emergency MRI in children had equal diagnostic accuracy to that in adults.
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Abscesso , Pescoço , Adolescente , Adulto , Dor no Peito , Criança , Humanos , Imageamento por Ressonância Magnética/métodos , Pescoço/diagnóstico por imagem , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
OBJECTIVES: Due to its superior soft-tissue contrast and ability to delineate abscesses, MRI has high diagnostic accuracy in neck infections. Whether MRI findings can predict the clinical course in these patients is unknown. The purpose of this study was to determine the clinical and prognostic significance of various MRI findings in emergency patients with acute neck infections. MATERIALS AND METHODS: We retrospectively reviewed the 3-T MRI findings of 371 patients with acute neck infections from a 5-year period in a single tertiary emergency radiology department. We correlated various MRI findings, including retropharyngeal (RPE) and mediastinal edema (ME) and abscess diameter, to clinical findings and outcomes, such as the need for intensive care unit (ICU) treatment and length of hospital stay (LOS). RESULTS: A total of 201 out of 371 patients (54%) with neck infections showed evidence of RPE, and 81 out of 314 patients (26%) had ME. Both RPE (OR = 9.5, p < 0.001) and ME (OR = 5.3, p < 0.001) were more prevalent among the patients who required ICU treatment than among those who did not. In a multivariate analysis, C-reactive protein (CRP) levels, RPE, and maximal abscess diameter were independent predictors of the need for ICU treatment, and CRP, ME, and maximal abscess diameter were independent predictors of LOS. CONCLUSION: In patients with an acute neck infection that requires emergency imaging, RPE, ME, and abscess diameter, as shown by MRI, are significant predictors of a more severe illness. KEY POINTS: ⢠Two hundred one out of 371 patients (54%) with neck infection showed evidence of retropharyngeal edema (RPE), and 81 out of 314 patients (26%) had mediastinal edema (ME). ⢠Maximal abscess diameter, RPE, and C-reactive protein (CRP) were independent predictors of the need for intensive care unit (ICU) treatment, and maximal abscess diameter, ME, and CRP were independent predictors of length of hospital stay. ⢠Prognostic significance of MRI findings was evident also while controlling for CRP values.
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Abscesso , Pescoço , Abscesso/diagnóstico por imagem , Humanos , Tempo de Internação , Imageamento por Ressonância Magnética , Pescoço/diagnóstico por imagem , Prognóstico , Estudos RetrospectivosRESUMO
OBJECTIVE: To analyse changes in patients' psychosocial well-being from before treatment until post-surgical orthodontic treatment (including retention) is completed. MATERIALS AND METHODS: Data was collected six times: before treatment (T0), 6-8 weeks after the placement of orthodontic appliances (T2), 3-4 weeks before surgery (T3), six weeks after surgery (T4), one year after surgery (T5) and after completing orthodontic treatment (T6; 20-57 months after surgery). At T0, 60 patients participated while at T6, data was available for 15 patients. All patients completed the Orthognathic Quality of Life Questionnaire (OQLQ), Rosenberg Self-Esteem Questionnaire (RSES), Acceptance and Action Questionnaire II (AAQ-II) and the Symptom Checklist 90 (SCL-90). All pairwise comparisons between variables were conducted with the Wilcoxon signed-rank test. RESULTS: OQLQ function, RSES, AAQ-II and SCL GSI worsened from T0 to T2. At T5, improvements compared to T0 were found in all aspects of OQLQ and SCL GSI. When comparing results at T6 to T0, improvements where only found in OQLQ sum, OQLQ facial aesthetics and OQLQ function. CONCLUSIONS: Although well-being of orthognathic patients seems to improve during treatment, many improvements cannot be verified anymore at the completion of the retention period. Most stable changes are found in the oral function component and in the facial aesthetics component of the OQLQ.
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Procedimentos Cirúrgicos Ortognáticos , Qualidade de Vida , Humanos , Aparelhos Ortodônticos , Procedimentos Cirúrgicos Ortognáticos/psicologia , Autoimagem , Inquéritos e QuestionáriosRESUMO
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.
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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.