Highly Sensitive Immunoassay for Long Forms of Cardiac Troponin T Using Upconversion Luminescence.

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 (0.40 ng/L) and 14-fold lower limit of quantitation (1.79 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.

Spectrally separated dual-label upconversion luminescence lateral flow assay for cancer-specific STn-glycosylation in CA125 and CA15-3.

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.

Improved sensitivity and automation of a multi-step upconversion lateral flow immunoassay using a 3D-printed actuation mechanism.

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.

Complement C1q in plasma induces nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticle antibody conjugates.

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.

Supersensitive photon upconversion based immunoassay for detection of cardiac troponin I in human plasma.

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.

Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin.

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL-1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals.