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.

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.

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.

Understanding FRET in Upconversion Nanoparticle Nucleic Acid Biosensors.

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 ∼24 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.

What Digital Immunoassays Can Learn from Ambient Analyte Theory: A Perspective.

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.

Engineering the Compositional Architecture of Core-Shell Upconverting Lanthanide-Doped Nanoparticles for Optimal Luminescent Donor in Resonance Energy Transfer: The Effects of Energy Migration and Storage.

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.

Clinical and prognostic significance of emergency MRI findings in neck infections.

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.

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.

Magnetic resonance imaging findings in pediatric neck infections-a comparison with adult patients.

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.

Does orthognathic treatment improve patients' psychosocial well-being?

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.

Frequency Encoding of Upconversion Nanoparticle Emission for Multiplexed Imaging of Spectrally and Spatially Overlapping Lanthanide Ions.

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.

Emergency neck MRI: feasibility and diagnostic accuracy in cases of neck infection.

BACKGROUND: Computed tomography (CT) has traditionally been the first-line imaging method in neck emergencies such as deep neck infections. Due to superior soft-tissue contrast, magnetic resonance imaging (MRI) may be an alternative to CT, also in emergency situations. PURPOSE: To characterize the use of routine MRI in neck emergencies, with an emphasis on clinical feasibility and diagnostic accuracy in cases of neck infection. MATERIAL AND METHODS: We conducted a retrospective cohort study of all primary neck MRI scans performed using a 3-T MRI device during a five-year follow-up period in a tertiary emergency radiology department. Imaging data were compared with final clinical diagnosis and surgical findings as reference standards. RESULTS: The search identified 461 primary neck MRI scans, of which 334 (72%) were performed on the basis of clinical suspicion of infection. Radiological evidence of infection was observed in 95% of these scans, and at least one abscess was detected in 229 cases (72% of confirmed infection). MRI had an overall technical success rate of 95% and had high positive predictive value for both infection (0.98) and detection of abscess (0.95). CONCLUSION: We found that emergency neck MRI can be successfully performed on most patients, and that MRI detects neck infection with a high accuracy. These results suggest that MRI may be an alternative to CT as the first or only imaging modality in neck emergencies.

Deep neck space infections: an upward trend and changing characteristics.

PURPOSE: This study reviews our experience with deep neck space infections (DNIs) requiring surgical intervention, including cervical necrotizing fasciitis. The aim of the study was to identify predisposing and aggravating factors of the disease and recognize the possible factors that can lead to life-threatening complications and slow down the healing process. METHODS: We compare the results to previous data from 1985 to 2005 to find possible alterations and changing trends. The characteristics of four lethal cases are described. This retrospective analysis includes patient data from 2004 to 2015 in tertiary referral hospital and in total, 277 patients were found. RESULTS: Surgical drainage through a neck opening ± intraoral incision was made in 215 (77.6%) patients, an intraoral incision was only made in 62 patients (22.4%). ICU care was needed in 66 (23.8%) cases. Odontogenic etiology (44.8%) was the most common origin. The most common comorbidity was a psychiatric disorder and/or dementia and occurred in 55 (19.9%) patients. Patients with underlying illnesses were more likely to be admitted to the ICU (p = 0.020), required a longer ICU stay (p = 0.004) and repeated surgery (p = 0.009). Gas formation seemed to be predictive of a more severe course of infection. Early extraction of the odontogenic foci was related to a lower length of stay (LOS) (p = 0.039). CONCLUSION: The annual numbers have risen from 14 to 24 cases per year when compared to previous data. DNIs remain a cause of lethal complications; the mortality was 1.4% and overall complications occurred in 61 (22.0%) patients.

Large-Scale Purification of Photon-Upconversion Nanoparticles by Gel Electrophoresis for Analogue and Digital Bioassays.

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).

Photochemical Ligation to Ultrasensitive DNA Detection with Upconverting Nanoparticles.

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.

Upconversion Cross-Correlation Spectroscopy of a Sandwich Immunoassay.

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.

Effective Shielding of NaYF4:Yb3+,Er3+ Upconverting Nanoparticles in Aqueous Environments Using Layer-by-Layer Assembly.

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.

Improving the sensitivity of immunoassays by reducing non-specific binding of poly(acrylic acid) coated upconverting nanoparticles by adding free poly(acrylic acid).

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.

Ratiometric Sensing and Imaging of Intracellular pH Using Polyethylenimine-Coated Photon Upconversion Nanoprobes.

Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models.

Lateral flow immunoassay with upconverting nanoparticle-based detection for indirect measurement of interferon response by the level of MxA.

Myxovirus resistance protein A (MxA) is a biomarker of interferon-induced gene expression state involved in many viral infections and some autoimmune disorders. It has a variety of potential utilities in clinical diagnostics, including distinguishing between bacterial and viral infections. Currently, MxA-assays are used for monitoring of IFN-ß therapy in multiple sclerosis (MS) patients. As a proof-of-concept for rapid quantitative measurement of interferon response, a lateral flow immunoassay (LFIA) with upconverting nanoparticle (UCNP) reporters was developed and evaluated with clinical whole blood samples to assess the potential for a rapid and user-friendly quantitative assay for MxA, since the currently available rapid test for MxA (FebriDX) produces only qualitative result. The high detection sensitivity enabled by the UCNP reporter technology allowed the sample pre-treatment with dilution of whole blood into lysis buffer at a detectable analyte concentration. The assay can be done within 2 hr and the results correlate with the reference MxA-ELISA, which requires an overnight incubation. With 36 samples, R2 for linear regression was 0.86. The assay detected 96% of the IFN-ß responders with 89% specificity using a cut-off level of 100 µg/L for an elevated MxA-concentration. J. Med. Virol. 89:598-605, 2017. © 2016 Wiley Periodicals, Inc.