Quantitative and discriminative analysis of nucleic acid samples using luminometric nonspecific nanoparticle methods.

Homogeneous simple assays utilizing luminescence quenching and time-resolved luminescence resonance energy transfer (TR-LRET) were developed for the quantification of nucleic acids without sequence information. Nucleic acids prevent the adsorption of a protein to europium nanoparticles which is detected as a luminescence quenching of europium nanoparticles with a soluble quencher or as a decrease of TR-LRET from europium nanoparticles to the acceptor dye. Contrary to the existing methods based on fluorescent dye binding to nucleic acids, equal sensitivities for both single- (ssDNA) and double-stranded DNA (dsDNA) were measured and a detection limit of 60 pg was calculated for the quenching assay. The average coefficient of variation was 5% for the quenching assay and 8% for the TR-LRET assay. The TR-LRET assay was also combined with a nucleic acid dye selective to dsDNA in a single tube assay to measure the total concentration of DNA and the ratio of ssDNA and dsDNA in the mixture. To our knowledge, such a multiplexed assay is not accomplished with commercially available assays.

Evaluation of the TPX MRSA assay for the detection of methicillin-resistant Staphylococcus aureus.

The aim of this study was to evaluate a new type of assay for the phenotypic detection of methicillin-resistant Staphylococcus aureus (MRSA). The assay is based on a point-of-care compatible two-photon excitation fluorescence detection technology (TPX). A collection of 243 epidemic MRSA isolates was tested in addition to 138 sporadic MRSA and 101 negative control strains. The assay proved to be both sensitive (97.9%) and specific (94.1%) in the identification of MRSA, with adequate positive (98.4%) and negative (92.2%) predictive values. The time required for obtaining a positive test result was less than 14 h for 99.0% of the MRSA true-positive samples. After a test run, the selectively enriched reaction mixtures may be recovered and further studied by molecular or standard phenotypic methods. The main benefits of the TPX methodology include a simple assay procedure, low reagent consumption, and a high-throughput capacity.

Utilization of kinetically enhanced monovalent binding affinity by immunoassays based on multivalent nanoparticle-antibody bioconjugates.

The monovalent binding affinity of high binding site density nanoparticle-antibody bioconjugates is shown to exceed the intrinsic affinity of the original, monoclonal antibody. The nanoparticle-antibody bioconjugates were prepared by covalent coupling of antibodies to long-lifetime fluorescent, europium(III) chelate nanoparticles, 107 nm in diameter. Experiments were carried out in standard microtitration wells to determine solid-phase association and dissociation rate constants, nonspecific binding, and affinity constants of the various binding site density nanoparticle-antibody bioconjugates and the conventionally labeled monoclonal antibody. The affinity constant for monovalent binding of a high binding site density bioconjugate (5.4 x 10(10) M(-1)) was 8-fold higher than the intrinsic affinity of the antibody (6.6 x 10(9) M(-1)). The separately measured association (2.5 x 10(6) M(-1) s(-1)) and dissociation (3.7 x 10(-5) s(-1)) rate constants of the bioconjugate were 2-fold higher and 4-fold lower, respectively, compared to the antibody. The dependence of the association rate constant of the density of the binding sites enhanced the kinetics and the affinity of the high binding site density bioconjugates. The nanoparticle labels with high specific activity, low nonspecific binding, and enhanced binding affinity of the nanoparticle-antibody bioconjugates contribute to the design of the next generation immunoassays with extreme sensitivity.

Supersensitive time-resolved immunofluorometric assay of free prostate-specific antigen with nanoparticle label technology.

BACKGROUND: The extreme specific activity of the long-lifetime fluorescent europium(III) chelate nanoparticles and the enhanced monovalent binding affinity of multivalent nanoparticle-antibody bioconjugates are attractive for noncompetitive immunoassay. METHODS: We used a noncompetitive, two-step immunoassay design to measure free prostate-specific antigen (PSA). Europium(III) chelate nanoparticles (107 nm in diameter) were coated with a monoclonal anti-PSA antibody (intrinsic affinity, 6 x 10(9) L/mol). The nanoparticle-antibody bioconjugates had an average of 214 active binding sites per particle and a monovalent binding affinity of 7 x 10(10) L/mol. The assay was performed in a low-fluorescence microtitration well passively coated with an another monoclonal anti-PSA antibody (affinity, 2 x 10(10) L/mol), and the europium(III) fluorescence was measured directly from the bottom of the well by a standard time-resolved microtitration plate fluorometer. RESULTS: The detection limit (mean + 2 SD) was 0.040 ng/L (7.3 x 10(5) molecules/mL), and the dynamic detection range covered four orders of magnitude in a 3-h total assay time. The imprecision (CV) over the whole assay range was 2-10%. The detection limit of the assay was limited by the fractional nonspecific binding of the bioconjugate to the solid phase (0.05%), which was higher than the nonspecific binding of the original antibody (<0.01%). CONCLUSIONS: The sensitivity of the new assay is equal to that of the ambient-analyte, microspot immunoassay and will be improved by use of optimized, high binding-site density nanoparticle-antibody bioconjugates with reduced nonspecific binding and improved monovalent binding affinity.

Europium nanoparticles and time-resolved fluorescence for ultrasensitive detection of prostate-specific antigen.

BACKGROUND: Nanoparticle-based detection technologies have the potential to improve detection sensitivity in miniature as well as in conventional biochemical assays. We introduce a detection technology that relies on the use of europium(III) nanoparticles and time-resolved fluorometry to improve the detection limit of biochemical assays and to visualize individual molecules in a microtiter plate format. METHODS: Streptavidin was covalently coated on 107-nm nanoparticles containing >30 000 europium molecules entrapped with beta-diketones. In a model assay system, these nanoparticles were used to trace biotinylated prostate-specific antigen (PSA) in a microtiter plate format. RESULTS: The detection limit (mean + 3 SD of the zero calibrator) of biotinylated PSA was 0.38 ng/L, corresponding to 10 fmol/L or 60 zeptomoles (60 x 10(-21) moles) of PSA. Moreover, single nanoparticles, representing individual PSA molecules, were visualized in the same microtiter wells with a time-resolved fluorescence microscope using a x10 objective. Single nanoparticles, possessing high specific activity, were also detected in solution by a standard time-resolved plate fluorometer. CONCLUSIONS: The universal streptavidin-coated europium(III) nanoparticle label is suitable for detection of any biotinylated molecule either in solution or on a solid phase. The europium(III) nanoparticle labeling technology is applicable to many areas of modern biochemical analysis, such as immunochemical and multianalyte DNA-chip assays as well as histo- and cytochemistry to improve detection sensitivities.

Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence.

Prostate-specific antigen (PSA) was detected in microtitre wells coated with a PSA-specific antibody using biotinylated antibody and streptavidin-coated, highly fluorescent 107 nm nanoparticles, which contained more than 30000 europium ions entrapped by beta-diketones. PSA was monitored directly on the surface of a well without any additional enhancement step. The sensitivity of the assay was 1.6 ng/L, corresponding to 50 fmol/L or 250 zeptomoles (250 x 10(-21) mol/L) of PSA. The high specific activity and low non-specific binding of the streptavidin-coated nanoparticles improved the sensitivity of the PSA assay 100-fold compared to the conventional europium-labelled streptavidin tracer in the same assay format. Additionally, the streptavidin-coated nanoparticle label made very rapid assays possible, due to the high affinity of the streptavidin-biotin complex and a high number of binding sites available for tracing the biotinylated antibody on the surface. Due to the inherent problems of tracing analyte with a complex of biotinylated antibody and streptavidin-coated nanoparticles, the streptavidin-coated nanoparticles reacting with the surface-captured analyte and biotinylated antibody was favoured and factors influencing this are discussed. This universal labelling technology can be applied to detect any biotinylated molecule, either in solution or on a solid phase, in order to improve detection sensitivities in many areas of biochemical analysis, such as cyto- and histochemistry, multianalyte DNA-chip assays and single-particle assays.

Time-resolved fluorescence imaging for quantitative histochemistry using lanthanide chelates in nanoparticles and conjugated to monoclonal antibodies.

Tissue and cell examinations have a potential to produce extremely valuable information about antigen quantities in samples. Using currently available methods, a truly quantitative analysis is nearly impossible. We have previously shown that immunohistochemical (IHC) detection of prostate-specific antigen and human glandular kallikrein from prostatic tissue, together with time-resolved fluorescence imaging (TRFI), is a suitable method for obtaining quantitative data from biological samples and that the signal response is linear. In this paper we show that Eu-chelate containing particles in the nanometer range are suitable labels for quantitative IHC. Even single nanoparticle molecules can be detected by TRFI and the signals measured can be readily quantitated. The signal intensity correlates very well with the amount of bound label, and the use of nanoparticles could markedly improve the sensitivity of quantitative IHC methods. TRFI provides a powerful tool for providing quantitative data about antigens or transcripts in tissue sections or cultured cells. It is also of major importance in standardization and optimization of protocols for fixation and tissue preparation, including antigen retrieval methods.

Miniature single-particle immunoassay for prostate-specific antigen in serum using recombinant Fab fragments.

BACKGROUND: Quantitative, miniaturized nucleic acid assays and immunoassays can be developed with single microparticles, microfluorometric detection, and intrinsically fluorescent lanthanide chelates in a multiple assay format to decrease reagent consumption, cost, and assay time. We used recombinant Fab fragments to capture and detect free and total prostate-specific antigen (PSA) from serum in a submicroliter volume single-particle immunoassay. METHODS: Genetically engineered thiol-Fab or thiolated monoclonal antibodies (mAbs) were covalently attached onto uniformly sized 60-microm maleimide-activated microparticles. Free and total PSA were detected with europium- or terbium-labeled Fab fragments on a single microparticle using a microfluorometer in a time-resolved mode. RESULTS: The detection limit of the free- and total-PSA assays (mean + 3 SD of zero calibrator) was 0.35 microg/L, with a total volume of 330 nL per particle. An excellent correlation was found in microparticle and microtiter-well assays for 21 serum samples: slopes for free and total PSA were 1.06+/-0.03 and 1.03+/-0.02, respectively (S(y|x) = 0.084 and 0.057 microg/L), with intercepts of 0.013+/-0.018 and 0.013+/-0.017 microg/L (R>0.99). Furthermore, the particle-immobilized Fab fragment had a PSA binding capacity 1.5-fold higher than the intact mAb capacity on a single microparticle. Capacity, kinetics, and sensitivity of the Fab fragment and intact mAb assays in the microparticle and microtiter well formats are discussed. CONCLUSIONS: With site-specific (cysteine tail) covalent attachment of Fab fragments on a microparticle, subattomole amounts of PSA can be detected quantitatively.

Very rapid, ionic strength-dependent association and folding of a heterodimeric leucine zipper.

Leucine zippers (coiled coils) are dimerization motifs found in several DNA-binding transcription factors. A parallel leucine zipper composed of the acidic chain X1-EYQALEKEVAQLEAENX2-ALEKEVAQLEHEG-amide and the basic chain X1-EYQALKKKVAQLKAKNX2ALKKKVAQLKHKG-amide was designed to study the kinetics of folding of a heterodimeric leucine zipper and to investigate the role of electrostatic attraction between oppositely charged peptide chains to the folding reaction. Each peptide alone did not form a leucine zipper at ionic strength (mu) < 1 M because of electrostatic repulsion between like charges in a homodimer. Therefore, the formation of the heterodimeric leucine zipper could be investigated by simple mixing of acidic and basic chains. To monitor folding, a fluorescent label was located either at the N-terminus (X1 = fluorescein-GGG, X2 = Q) or in the center of the coiled coil (X1 = acetyl, X2 = W). Folding could be described by a simple two-state reaction involving the disordered monomers and the folded heterodimer. The same bimolecular rate constant (k(on)) was observed independent of the location of the fluorescent label, indicating that both fluorescence probes monitored the same reaction. Lowering of the ionic strength increased k(on) from 4 x 10(6) M-1 s-1 (mu = 525 mM) to about 5 x 10(7) M-1 s-1 (mu = 74 mM). When extrapolated to mu = O, k(on) was approximately 10(9) M-1 s-1, which is near the diffusion limit. In contrast, the rate of dissociation depended very weakly on ionic strength; k(off) decreased only by about 2-fold when mu was lowered from 525 to 74 mM. Equilibrium association constants (Ka) of the heterodimeric zippers measured directly and calculated from kinetic constants (Ka = k(on)/k(off) were in good agreement. The observed two-state mechanism, the strong dependence on ionic strength of k(on) but not of k(off), and the nearly diffusion-limited association rate at very low ionic strength point to a folding pathway in which the formation of an electrostatically stabilized dimeric intermediate may be rate-limiting and the subsequent folding to the final dimer is very rapid and follows a "down-hill" free energy landscape. The small increase of k(off) at increasing ionic strength indicates a minor contribution of electrostatics to the stability of the folded leucine zipper.