Switchable reporter enzymes based on mutually exclusive domain interactions allow antibody detection directly in solution

Detection of antibodies is essential for the diagnosis of many diseases including infections, allergies, and autoimmune diseases. Current heterogeneous immunoassays require multiple time-consuming binding and washing steps, which limits their application in point-of-care diagnostics and high-throughput screening. Here, we report switchable reporter enzymes that allow simple colorimetric detection of antibodies directly in solution. Our approach is based on the antibody-induced disruption of an intramolecular interaction between TEM1 ß-lactamase and its inhibitor protein BLIP. Using the HIV1-p17 antibody as an initial target, the interaction between enzyme and inhibitor was carefully tuned to yield a reporter enzyme whose activity increased 10-fold in the presence of pM antibody concentrations. Reporter enzymes for two other antibodies (HA-tag and Dengue virus type I) were obtained by simply replacing the epitope sequences. This new sensor design represents a modular and generic approach to construct antibody reporter enzymes without the cumbersome optimization required by previous engineering strategies

Monitoring bile acid transport in single living cells using a genetically encoded Förster resonance energy transfer sensor

Bile acids are pivotal for the absorption of dietary lipids and vitamins and function as important signaling molecules in metabolism. Here, we describe a genetically encoded fluorescent bile acid sensor (BAS) that allows for spatiotemporal monitoring of bile acid transport in single living cells. Changes in concentration of multiple physiological and pathophysiological bile acid species were detected as robust changes in Förster resonance energy transfer (FRET) in a range of cell types. Specific subcellular targeting of the sensor demonstrated rapid influx of bile acids into the cytoplasm and nucleus, but no FRET changes were observed in the peroxisomes. Furthermore, expression of the liver fatty acid binding protein reduced the availability of bile acids in the nucleus. The sensor allows for single cell visualization of uptake and accumulation of conjugated bile acids, mediated by the Na(+)-taurocholate cotransporting protein (NTCP). In addition, cyprinol sulphate uptake, mediated by the putative zebrafish homologue of the apical sodium bile acid transporter, was visualized using a sensor based on the zebrafish farnesoid X receptor. The reversible nature of the sensor also enabled measurements of bile acid efflux in living cells, and expression of the organic solute transporter αβ (OSTαβ) resulted in influx and efflux of conjugated chenodeoxycholic acid. Finally, combined visualization of bile acid uptake and fluorescent labeling of several NTCP variants indicated that the sensor can also be used to study the functional effect of patient mutations in genes affecting bile acid homeostasis. CONCLUSION: A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real tim

One-step homogeneous magnetic nanoparticle immunoassay for biomarker detection directly in blood plasma

Assay technologies capable of detecting low biomarker concentrations in complex biological samples are fundamental for biological research and for applications in medical diagnostics. In this paper we address the challenge to perform protein biomarker detection homogeneously in one single step, applying a minute amount of reagent directly into whole human blood plasma, avoiding any sample dilution, separation, amplification, or fluid manipulation steps. We describe a one-step homogeneous assay technology based on antibody-coated magnetic nanoparticles that are spiked in very small amount directly into blood plasma. Pulsed magnetic fields and a double-linker molecular architecture are used to generate high biomarker-induced binding and low nonspecific binding between the nanoparticles. We demonstrate dose–response curves for prostate specific antigen (PSA) measured in undiluted human blood plasma with a detection limit of 400–500 femtomol/L, in a total assay time of 14 min and an optically probed volume of only 1 nL. We explain the dose–response curves with a model based on discrete binding of biomarker molecules onto the nanoparticles, which allows us to extract reaction parameters for the binding of biomarker molecules onto the nanoparticles and for the biomarker-induced binding between nanoparticles. The demonstrated analytical performance and understanding of the nanoparticle assay technology render it of interest for a wide range of applications in quantitative biology and medical diagnostics

DNA-directed control of enzyme-inhibitor complex formation: a modular approach to reversibly switch enzyme activity

DNA-templated reversible assembly of an enzyme–inhibitor complex is presented as a new and highly modular approach to control enzyme activity. TEM1-ß-lactamase and its inhibitor protein BLIP were conjugated to different oligonucleotides, resulting in enzyme inhibition in the presence of template strand. Formation of a rigid dsDNA linker upon addition of a complementary target strand disrupts the enzyme–inhibitor complex and results in the restoration of enzyme activity, enabling detection of as little as 2 fmol DNA. The noncovalent assembly of the complex allows easy tuning of target and template strands without changing the oligonucleotide-functionalized enzyme and inhibitor domains. Using a panel of eight different template sequences, restoration of enzyme activity was only observed in the presence of the target viral DNA sequence. The use of stable, well-characterized protein domains and the intrinsic modularity of our system should allow easy integration with DNA/RNA-based logic circuits for applications in biomedicine and molecular diagnostics