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Nucleic acid diagnostics is a growing sector in life sciences. Numerous applications based on the amplification of nucleic acids are utilized to determine the presence, composition, quality, or quantity of the sequence in question. However, amplification is a time-consuming, laborious, and error-prone process. Therefore, it would be preferable to perform tests with a minimized number of amplification cycles or, alternatively, completely without the amplification. We present two very sensitive sandwich assay concepts for a measurement of nucleic acids. The assays are performed either in standard microtitration wells or on microparticles coated with capture probes. Detection-probe-coated europium(III) nanoparticle labels are used for signal generation. The detection limits of the microtitration well and microparticle applications were 4.0 × 105 and 6.1 × 104 copies of target sequence, respectively. The reference assay, based on the detection of europium(III) chelate-labelled detection probes, had a detection limit of 8.5 × 107 copies. Thus, 100–1000-fold improvement in the sensitivity was achieved. The high sensitivity of the designed assays is based on the long-lifetime fluorescence of nanoparticle labels, on time-resolved fluorometry, and on signal rather than target amplification. The presented sandwich assays provide the tool for a development of direct, amplification-free detection of nucleic acids.

Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications. We investigated the possibility of using europium(III)-labeled 68-nm nanoparticles coated with monoclonal antibodies or streptavidin (SA) to detect prostate-specific antigen (PSA) in serum. The selection of a suitable antibody pair and interference caused by the combination of nanoparticle label and structurally complex analyte were of special interest. A set of antibodies recognizing different epitope areas of PSA was mapped to find the optimal antibody pair for the immunometric nanoparticle-based assay. Different assay configurations were tested to obtain a good correlation with a conventional method based on biotinylated detection antibodies and europium(III) chelate-labeled streptavidin. Monoclonal capture antibody 5E4 was covalently coated on a microtitration well surface; biotinylated 5H6 monoclonal antibody (Mab) was used for detection, and europium(III)-labeled streptavidin-coated nanoparticles were utilized for signal generation. Total PSA concentrations were determined from a panel of male serum samples to test the developed assay. The correlation of the nanoparticle-based and reference assays was good; y=0.9844x−0.1252, R2=0.98, n=27; and the lowest limit of detection of the assay (LLD=0.83 ng/l) was 35-fold lower than for the reference method. The assay application presented here, where a structurally complex analyte is detected, combines the exceptionally high affinity of streptavidin–biotin technology and the high specific activity of long lifetime fluorescence nanoparticle labels. The general characteristics of this combination should permit the development of various immunoassay applications featuring high sensitivity, rapidity, and low consumption of reagents.

The present study describes the development of a quantitative miniaturized single microparticle immunoassay. The main objective of the study was to evaluate the performance of a miniature heterogeneous immunoassay on a single microparticle in respect to assay kinetics, volume, and sensitivity, binding capacity of microparticles and sensitivity using europium(III) nanoparticle labels. The performance of the single microparticle assay of prostate-specific antigen (PSA) was investigated using different-sized microparticles (60–920 μm in diameter) and microtiter well as a solid-phase. Equilibration time of the assay was shown to be dependent in a linear manner on surface-to-volume ratio, i.e. larger surface-to-volume translated to a faster reaction. However, no correlation between PSA binding capacity and equilibration time was observed in these kinetic studies. Only moderate improvement in assay kinetics was found when PSA binding capacity was increased on a microparticle. Using europium(III) nanoparticle labels, 107 nm in diameter, coated with streptavidin a detection sensitivity of 30 ng l −1 (0.1 amol) was achieved in 1 μl total assay volume per microparticle. This was 50-fold higher compared to the same assay performed with intrinsically fluorescent europium(III) labels.

Background : Recent proceedings in utilization of europium(III) chelate-dyed polystyrene nanoparticles as labels have combined the advantages of an enhanced monovalent binding affinity and a high specific activity of nanoparticle–antibody bioconjugate. Our objective was to evaluate the performance of the nanoparticle label technology with biological samples in an immunoassay of free prostate-specific antigen (PSA-F) using a standard microtitration well platform. Methods : Long-lifetime luminescent europium(III)-chelate nanoparticles, 107 nm in diameter, were coated with a PSA-F specific monoclonal antibody. The two-step noncompetitive immunoassay was performed in a microtitration well coated with a second monoclonal antibody. The signal of the surface-bound nanoparticle–antibody bioconjugates was measured directly from the bottom of the well using a standard time-resolved plate fluorometer. Results : The detection limit (mean+2SD) of the nanoparticle-based PSA-F assay was 0.21 ng/l using a 20-μl sample volume. The assay response was linear up to 5 μg/l, and the functional sensitivity was approximately 0.5 ng/l. The within-run imprecision for spiked serum samples at concentrations 0.0005–0.5 μg/l was 6.4–21.8%, and the within-run and between-run imprecisions for serum samples at concentrations 0.2–2.5 μg/l were 3.4–7.2% and 4.4–7.6%, respectively. The concentrations obtained from serum samples correlated well with the reference immunoassay; slope=1.018±0.018; intercept=0.012±0.021 μg/l; S y | x =0.112 μg/l; r =0.993; n =51. Conclusions : The developed method demonstrated acceptable performance characteristics allowing clinical studies utilizing patient samples with extremely low concentrations of PSA-F. The present assay detected PSA-F in most of samples from prostatectomized men and in few samples from healthy women that were nondetectable according to the reference immunoassay.

Long-lifetime fluorescent europium(III) chelate nanoparticles have been shown to be applicable as labels in various heterogeneous and homogeneous immunoassays. Assay performance can be improved when these intrinsically labeled nanoparticles are used in combination with time-resolved fluorescence detection. In several previous publications, we have shown the potential of nanoparticle labels in different assay applications. In heterogeneous assays, the dynamic range of assays at low concentrations can be extended (1)(2)(3). Furthermore, the kinetic characteristics of assays can be improved by use of detection antibody-coated high-specific-activity nanoparticle labels instead of conventionally labeled detection antibodies (4). In homogeneous assays, europium(III) nanoparticles have been shown to be efficient donors in fluorescence resonance energy transfer, enabling simple and rapid high-throughput screening (5). Heterogeneous and homogeneous nanoparticle-label-based assays can be run with various sample matrixes, e.g., serum (3), heparin plasma (3), and mucus (S. Huopalahti, A. Valanne, T. Soukka, R. Vainionpaa, T. Lovgren, and H. Harma, University of Turku, unpublished data). In the present work we describe a kinetic, heterogeneous, one-step immunoassay application in which europium(III) nanoparticle labels are used in an all-in-one dry-reagent format (Aio!™) (6) to produce a simple, rapid, and sensitive sandwich-type assay for thyroid-stimulating hormone (TSH). Conventionally, the Aio! technology is based on the europium(III) chelate label, but in the present …