Single-molecule microfluidic assay for prostate-specific antigen based on magnetic beads and upconversion nanoparticles.

Early-stage diagnosis of prostatic carcinoma is essential for successful treatment and, thus, significant prognosis improvement. In laboratory practice, the standard non-invasive diagnostic approach is the immunochemical detection of the associated biomarker, prostate-specific antigen (PSA). Ultrasensitive detection of PSA is essential for both diagnostic and recurrence monitoring purposes. To achieve exceptional sensitivity, we have developed a microfluidic device with a flow-through cell for single-molecule analysis using photon-upconversion nanoparticles (UCNPs) as a detection label. For this purpose, magnetic microparticles (MBs) were first optimized for the capture and preconcentration of PSA and then used to implement a bead-based upconversion-linked immunoassay (ULISA) in the microfluidic device. The digital readout based on counting single nanoparticle-labeled PSA molecules on MBs enabled a detection limit of 1.04 pg mL-1 (36 fM) in 50% fetal bovine serum, which is an 11-fold improvement over the respective analog MB-based ULISA. The microfluidic technique conferred several other advantages, such as easy implementation and the potential for achieving high-throughput analysis. Finally, it was proven that the microfluidic setup is suitable for clinical sample analysis, showing a good correlation with a reference electrochemiluminescence assay (recovery rates between 97% and 105%).

Artificial Intelligence-Aided Massively Parallel Spectroscopy of Freely Diffusing Nanoscale Entities.

Massively parallel spectroscopy (MPS) of many single nanoparticles in an aqueous dispersion is reported. As a model system, bioconjugated photon-upconversion nanoparticles (UCNPs) with a near-infrared excitation are prepared. The UCNPs are doped either with Tm3+ (emission 450 and 802 nm) or Er3+ (emission 554 and 660 nm). These UCNPs are conjugated to biotinylated bovine serum albumin (Tm3+-doped) or streptavidin (Er3+-doped). MPS is correlated with an ensemble spectra measurement, and the limit of detection (1.6 fmol L-1) and the linearity range (4.8 fmol L-1 to 40 pmol L-1) for bioconjugated UCNPs are estimated. MPS is used for observing the bioaffinity clustering of bioconjugated UCNPs. This observation is correlated with a native electrophoresis and bioaffinity assay on a microtiter plate. A competitive MPS bioaffinity assay for biotin is developed and characterized with a limit of detection of 6.6 nmol L-1. MPS from complex biological matrices (cell cultivation medium) is performed without increasing background. The compatibility with polydimethylsiloxane microfluidics is proven by recording MPS from a 30 µm deep microfluidic channel.

Absolute Counting Method with Multiplexing Capability for Estimating the Number Concentration of Nanoparticles Using Anisotropically Collapsed Gels.

Number concentration─the number of nanoparticles in a given volume─is an important characteristic of any nanoparticle dispersion. However, its estimation for small nanoparticles (∼30 nm) is generally challenging. We introduce an absolute and widely applicable method for analyzing aqueous dispersions of nanoparticles. An innovative immobilization of nanomaterials in the anisotropically collapsed agarose gel is pioneered, followed by optical microscopy and nanoparticle counting. The number of counted nanoparticles is inherently coupled with sampled volume (517 pL) and translates to the number concentration. Photon-upconversion, fluorescence, bright-field, and dark-field microscopy techniques have been proven applicable and used for imaging lanthanide-doped photon-upconversion nanoparticles, their bioconjugates with antibodies, silica dye-doped fluorescent nanoparticles, quantum dots, and pure silica submicron particles. The precision and linearity were characterized by constructing a dilution series of photon-upconversion nanoparticles. The limit of detection was 2.0 × 106 mL-1, and the working range was from 4.4 × 107 to 2.2 × 1010 mL-1. The quantification of nanoparticle clusters was achieved by a thorough analysis of the micrographs. The accuracy was confirmed using gravimetric analysis and transmission electron microscopy as a reference. Multiplexed detection of two nanoparticle types in a mixed dispersion was feasibly demonstrated. The low thickness of the collapsed gel (<1 µm) supported extremely sensitive imaging. This was proven by imaging Tm3+-doped photon-upconversion nanoparticles (17 nm hydrodynamic diameter) with a nanoparticle emission rate of only ∼900 photons/s at a wavelength of 800 nm (excitation wavelength 976 nm).

Seroprevalence of Measles Antibodies in the Population of the Olomouc Region, Czech Republic-Comparison of the Results of Four Laboratories.

OBJECTIVES: Although the incidence of measles has decreased globally since the introduction of regular vaccination, its frequency has increased again in recent years. The study is focused on data from the Olomouc Region in the Czech Republic analyzed in four laboratories. The obtained results were compared with already published data. METHODS: The data were provided by individual laboratories in an anonymized form-age at the time of the examination, sex, and result of test. Samples were collected between June 2018 and September 2019 and evaluated on the scale positive-borderline-negative. RESULTS: A total of 7962 sera samples were evaluated using three different methods-two types of ELISA tests and CLIA. Positive result was issued in a total of 62.6 percent of samples, but the results of individual laboratories varied widely from 55.5 to 70.8 percent. However, the same trend with the highest levels of antibodies in people born before beginning of vaccination was observed. CONCLUSIONS: Data show significantly different results depending on the individual laboratories and the detection kits used. The underestimation of the proportion of positive results can cause problems in selecting individuals for revaccination with a live vaccine, which may fail in weakly positive individuals.

Photon-upconversion barcode for monitoring an enzymatic reaction with a fluorescence reporter in droplet microfluidics.

We report luminescent photon-upconversion barcodes for indexing the chemical content of droplets. The barcode is compatible with the simultaneous detection of fluorescence. The encoding and decoding of the initial concentration of enzyme ß-galactosidase and substrate 4-methylumbelliferyl ß-d-galactopyranoside are described. The fluorescent product 4-methylumbelliferone is detected simultaneously with the barcode.

Photon-Upconversion Barcoding with Multiple Barcode Channels: Application for Droplet Microfluidics.

Barcoding facilitates high-throughput analytical methods in complex matrixes with a reduced volume of sample, reagents, time, and cost. Because of orthogonality to fluorescence, photon-upconversion barcodes attracted considerable attention in recent years. We constructed an epiluminescence detector, which, for the first time, demonstrated the reading of photon-upconversion spectra from microdroplets in a microfluidic chip with frequency up to 10 Hz. Non-negative least-squares deconvolution enabled the reading of an unprecedented number of photon-upconversion barcode channels (six) from emission spectra (excitation 980 nm, emission 430-875 nm). The standard deviation of barcode reading from microdroplets was ∼1%. Described barcoding can be, for example, used for multiparameter titrations, multiplexed biological and chemical assays, optimizations on a microfluidic platform, and preparation of barcoded concentration gradients and libraries.