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 β-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 × 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 ×10 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.

We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assay. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthetized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.

In this article, we report on the formation and mode-of-operation of an affinity biosensor, where alternate layers of biotin/streptavidin/biotinylated-CRP-antigen/anti-CRP antibody are grown on printed gold electrodes on disposable paper-substrates. We have successfully demonstrated and detected the formation of consecutive layers of supra-molecular protein assembly using an electrical (impedimetric) technique. The formation process is also supplemented and verified using conventional surface plasmon resonance (SPR) measurements and surface sensitive characterization techniques, such as X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The article provides a possible biosensor development scheme, where—(1) fabrication of paper substrate (2) synthesis of gold nanoparticle inks (3) inkjet printing of gold electrodes on paper (4) formation of the biorecognition layers on the gold electrodes and (5) electrical (impedimetric) analysis of growth—all are coupled together to form a test-structure for a recyclable and inexpensive point-of-care diagnostic platform.

In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β₂-adrenoreceptor (β₂AR) antagonists and agonists in intact human embryonic kidney HEK293_i cells overexpressing human β₂-adrenergic receptors. Two antagonists (propranolol, alprenolol) and 2 agonists (metaproterenol, terbutaline) for β₂AR were investigated in a displacement assay using europium(III)-labeled pindolol ligand. The assay Z′ values ranged from 0.68 to 0.78, the coefficient of variation was less than 10%, and the K_i values were 19 nM for propranolol and alprenolol and 14 and 5.9 µM for metaproterenol and terbutaline, respectively. The QRET technique with β₂AR was also applied to LOPAC compound library screening, yielding nearly error-free recognition of known binders. This simple and cost-effective technique can be readily adapted to laboratory and industrial-scale screening. (Journal of Biomolecular Screening 2009:936-943)

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein’s physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a ‘Gold Standard’ for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.