Interactions of 17ß-Hydroxysteroid Dehydrogenase Type 10 and Cyclophilin D in Alzheimer's Disease.

The nucleus-encoded 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid ß peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17ß-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17ß-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (ka 2.0 × 105 M1s-1, kd 5.8 × 104 s-1, and KD 3.5 × 10-10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17ß-HSD10-cypD complexes were decreased and those of total amyloid ß increased. Moreover, the levels of 17ß-HSD10-cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17ß-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid ß. Levels of 17ß-HSD10-cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

Ionic Environment Affects Biomolecular Interactions of Amyloid-ß: SPR Biosensor Study.

In early stages of Alzheimer's disease (AD), amyloid beta (Aß) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17ß-hydroxysteroid dehydrogenase 10 (17ß-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aß affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aß (Aß1-40, Aß1-42) with cypD and 17ß-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K+ and Mg2+ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aß1-40 and Aß1-42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aß1-40 to cypD and 17ß-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K+ and increased concentrations of Mg2+ promote the interaction of both mitochondrial proteins with Aß1-42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.

Hsp70 Trap Assay for Detection of Misfolded Subproteome Related to Myelodysplastic Syndromes.

The onset and progression of numerous serious diseases (e.g., various types of malignancies, neurodegenerative diseases, and cardiac diseases) are, on a molecular level, associated with protein modifications and misfolding. Current methods for the detection of misfolded proteins are not able to detect the whole misfolded subproteome and, moreover, are rather laborious and time consuming. Herein, we report on a novel simple method for the detection of misfolded proteins employing a surface plasmon resonance (SPR) biosensor and heat shock protein 70 (Hsp70) that recognizes and traps misfolded proteins in a nucleotide-dependent manner. We use this method for the detection of misfolded proteins in blood plasma of patients with various subtypes of myelodysplastic syndromes (MDS) and healthy donors. Our results reveal significantly elevated levels of misfolded proteins in the two stages of MDS that are most affected by oxidative stress: low-risk (RARS) and intermediate-risk (RCMD) patients. This approach can be extended to a variety of diseases and provides unique insights into the thus far unexplored area of blood proteome.

High-performance biosensor exploiting a light guidance in sparse arrays of metal nanoparticles.

We introduce a new approach to plasmonic biosensing with superior biosensing properties based on spectroscopy of an electromagnetic mode guided by a monolayer of sparsely distributed colloidal plasmonic nanoparticles. The theoretical prediction of optical and sensing performance is confirmed by an experimental study in which adsorption of biomolecules on the sensor surface is studied. An unprecedentedly high figure of merit related to surface refractive index changes (FOMS) is demonstrated for distances of the biomolecules from the sensor surface up to 30 nm, which makes this approach a promising candidate for localized biosensing.

Nanoplasmonic Ruler for Measuring Separation Distance between Supported Lipid Bilayers and Oxide Surfaces.

Unraveling the details of how supported lipid bilayers (SLBs) are coupled to oxide surfaces is experimentally challenging, and there is an outstanding need to develop highly surface-sensitive measurement strategies to determine SLB separation distances. Indeed, subtle variations in separation distance can be associated with significant differences in bilayer-substrate interaction energy. Herein, we report a nanoplasmonic ruler strategy to measure the absolute separation distance between SLBs and oxide surfaces. A localized surface plasmon resonance (LSPR) sensor was employed to track SLB formation onto titania- and silica-coated gold nanodisk arrays. To interpret measurement data, an analytical model relating the LSPR measurement response to bilayer-substrate separation distance was developed based on finite-difference time-domain (FDTD) simulations and theoretical calculations. The results indicate that there is a larger separation distance between SLBs and titania surfaces than silica surfaces, and the trend was consistent across three tested lipid compositions. We discuss these findings within the context of the interfacial forces underpinning bilayer-substrate interactions, and the nanoplasmonic ruler strategy provides the first direct experimental evidence comparing SLB separation distances on titania and silica surfaces.

Microfluidic Analyte Transport to Nanorods for Photonic and Electrochemical Sensing Applications.

There has recently been a growing use of surface bound nanorods within electrochemical and optical sensing applications. Predictions of the microfluidic rate of analyte transport to such nanorods (either individual or to an array) remain important for sensor design and data analysis; however, such predictions are difficult, as nanorod aspect ratios can vary by several orders of magnitude. In this study, through the use of numerical simulation, we propose an explicit analytical approach to predict the steady-state diffusion-limited rate of mass transport to (individual) surface bound nanorods of variable aspect ratio. We show that, when compared to simulation, this approach provides accurate estimations across a wide range of Péclet numbers.

Ultralow-Fouling Behavior of Biorecognition Coatings Based on Carboxy-Functional Brushes of Zwitterionic Homo- and Copolymers in Blood Plasma: Functionalization Matters.

Fouling from complex biological fluids such as blood plasma to biorecognition element (BRE)-functionalized coatings hampers the use of affinity biosensor technologies in medical diagnostics. Here, we report the effects the molecular mechanisms involved in functionalization of low-fouling carboxy-functional coatings have on the BRE capacity and resistance to fouling from blood plasma. The specific mechanisms of EDC/NHS activation of carboxy groups, BRE attachment, and deactivation of residual activated groups on recently developed ultra-low-fouling carboxybetaine polymer and copolymer brushes (pCB) as well as conventional carboxy-terminated oligo(ethylene glycol)-based alkanethiolate self-assembled monolayers (OEG-SAMs) are studied using the polarization modulation infrared reflection/absorption spectroscopy, X-ray photoelectron spectroscopy, and surface plasmon resonance methods. It is shown that the fouling resistance of BRE-functionalized pCB coatings is strongly influenced by a deactivation method affecting the ultra-low-fouling molecular structure of the brush and surface charges. It is revealed that, in contrast to free carboxy-group-terminated OEG-SAMs, only a partial deactivation of EDC/NHS-activated zwitterionic carboxy groups by spontaneous hydrolysis is possible in the pCB brushes. The fouling resistance of activated/BRE-functionalized pCB is shown to be recovered only by covalent attachment of amino acid deactivation agents to residual activated carboxy groups of pCB. The developed deactivation procedure is further combined with ultra-low-fouling brushes of random copolymer carboxybetaine methacrylamide (CBMAA) and N-(2-hydroxypropyl) methacrylamide (HPMAA) with optimized CBMAA content (15%) providing a BRE-functionalized coating with superior fouling resistance over various carboxy-functional low-fouling coatings including homopolymer pCB brushes and OEG-SAMs. The biorecognition capabilities of pHPMAA-CBMAA(15%) are demonstrated via the sensitive label-free detection of a microRNA cancer biomarker (miR-16) in blood plasma.

Functional gold nanoparticles for optical affinity biosensing.

Functional gold nanoparticles (AuNPs) are commonly used to enhance the response of optical affinity biosensors. In this work, we investigated the effect of preparation conditions on functional properties of AuNPs functionalized with antibody (Ab-AuNPs), specifically AuNPs with antibody against carcinoembryonic antigen (CEA) covalently attached via carboxy-terminated oligo-ethylene thiolate linker layer. The following parameters of preparation of Ab-AuNP have been found to have a significant effect on Ab-AuNP performance in affinity biosensors: the time of reaction of activated AuNPs with antibody, concentrations of antibody and amino-coupling reagents, and composition of immobilization buffer (molarity and salt content). In contrast, pH of immobilization buffer has been demonstrated to have only a minor influence. Our experiments showed that the Ab-AuNPs prepared under optimum conditions offered a binding efficiency of Ab-AuNPs to CEA as high as 63%, which is more than 4 times better than the best efficiencies reported for similar functional AuNPs so far. We employed these Ab-AuNPs with a surface plasmon resonance (SPR) biosensor for the detection of CEA and showed that the Ab-AuNPs enhanced the sensor response to CEA by a factor of 1000. We also demonstrated that the Ab-AuNPs allow the biosensor to detect CEA at concentrations as low as 12 and 40 pg/mL in buffer and 50% blood plasma, respectively.

Pregnancy-Associated Plasma Protein A2 in Hemodialysis Patients: Significance for Prognosis.

BACKGROUND: Pregnancy-associated plasma protein A (PAPP-A) is associated with adverse outcome of long-term hemodialysis patients (HD). The aim of the study was to test whether its homolog pregnancy-associated plasma protein A2 (PAPP-A2) can be detected in serum of HD patients and to define its significance. METHODS: The studied group consisted of 102 long-term HD patients and 25 healthy controls. HD patients were prospectively followed up for five years (2009-2014). PAPP-A2 was measured by surface plasmon resonance biosensor, PAPP-A by time resolved amplified cryptate emission. RESULTS: PAPP-A2, similarly as PAPP-A, was significantly increased in HD patients (median (interquartile range)) PAPP-A2: 6.2 (2.6-10.8) ng/mL, vs. 3.0 (0.7-5.9) ng/mL, p=0.006; PAPP-A: 18.9 (14.3-23.4) mIU/L, vs. 9.5 (8.4-10.5) mIU/L, p<0.001). In HD patients, PAPP-A2 correlated weakly but significantly with PAPP-A (τ=0.193, p=0.004). Unlike PAPP-A, PAPP-A2 was not significant for prognosis of HD patients when tested alone. There was a significant interaction between PAPP-A and PAPP-A2 on the mortality due to infection of HD patients (p=0.008). If PAPP-A was below median, mortality due to infection was significantly higher for patients with PAPP-A2 values above median than for patients with low PAPP-A2 levels (p=0.011). CONCLUSION: PAPP-A2 is increased in HD patients and interacts with PAPP-A on patients´ prognosis.

(Bio)Sensing Using Nanoparticle Arrays: On the Effect of Analyte Transport on Sensitivity.

There has recently been an extensive amount of work regarding the development of optical, electrical, and mechanical (bio)sensors employing planar arrays of surface-bound nanoparticles. The sensor output for these systems is dependent on the rate at which analyte is transported to, and interacts with, each nanoparticle in the array. There has so far been little discussion on the relationship between the design parameters of an array and the interplay of convection, diffusion, and reaction. Moreover, current methods providing such information require extensive computational simulation. Here we demonstrate that the rate of analyte transport to a nanoparticle array can be quantified analytically. We show that such rates are bound by both the rate to a single NP and that to a planar surface (having equivalent size as the array), with the specific rate determined by the fill fraction: the ratio between the total surface area used for biomolecular capture with respect to the entire sensing area. We characterize analyte transport to arrays with respect to changes in numerous parameters relevant to experiment, including variation of the nanoparticle shape and size, packing density, flow conditions, and analyte diffusivity. We also explore how analyte capture is dependent on the kinetic parameters related to an affinity-based biosensor, and furthermore, we classify the conditions under which the array might be diffusion- or reaction-limited. The results obtained herein are applicable toward the design and optimization of all (bio)sensors based on nanoparticle arrays.

Copolymer Brush-Based Ultralow-Fouling Biorecognition Surface Platform for Food Safety.

Functional polymer coatings that combine the ability to resist nonspecific fouling from complex media with high biorecognition element (BRE) immobilization capacity represent an emerging class of new functional materials for a number of bioanalytical and biosensor technologies for medical diagnostics, security, and food safety. Here, we report on a random copolymer brush surface - poly(CBMAA-ran-HPMAA) - providing high BRE immobilization capacity while simultaneously exhibiting ultralow-fouling behavior in complex food media. We demonstrate that both the functionalization and fouling resistance capabilities of such copolymer brushes can be tuned by changing the surface contents of the two monomer units: nonionic N-(2-hydroxypropyl) methacrylamide (HPMAA) and carboxy-functional zwitterionic carboxybetaine methacrylamide (CBMAA). It is demonstrated that the resistance to fouling decreases with the surface content of CBMAA; poly(CBMAA-ran-HPMAA) brushes with CBMAA molar content up to 15 mol % maintain excellent resistance to fouling from a variety of homogenized foods (hamburger, cucumber, milk, and lettuce) even after covalent attachment of BREs to carboxy groups of CBMAA. The poly(CBMAA 15 mol %-ran-HPMAA) brushes functionalized with antibodies are demonstrated to exhibit fouling resistance from food samples by up to 3 orders of magnitude better when compared with the widely used low-fouling carboxy-functional oligo(ethylene glycol) (OEG)-based alkanethiolate self-assembled monolayers (AT SAMs) and, furthermore, by up to 2 orders of magnitude better when compared with the most successful ultralow-fouling biorecognition coatings - poly(carboxybetaine acrylamide), poly(CBAA). When model SPR detections of food-borne bacterial pathogens in homogenized foods are used, it is also demonstrated that the antibody-functionalized poly(CBMAA 15 mol %-ran-HPMAA) brush exhibits superior biorecognition properties over the poly(CBAA).

Surface plasmon resonance biosensor for detection of pregnancy associated plasma protein A2 in clinical samples.

Pregnancy associated plasma protein A2 (PAPP-A2) is a metalloproteinase that plays multiple roles in fetal development and post-natal growth. Here we present a novel surface plasmon resonance (SPR) biosensor for the rapid and quantitative detection of PAPP-A2 in blood samples. This biosensor uses a single surface referencing approach and a sandwich assay with functionalized gold nanoparticles for signal enhancement. We demonstrate that this SPR biosensor enables the detection of PAPP-A2 in 30 % blood plasma at levels as low as 3.6 ng/mL. We also characterize the performance of the biosensor and evaluate its cross-reactivity to a PAPP-A analogue. Finally, we utilize this SPR biosensor for the detection of PAPP-A2 in blood serum from two groups of subjects: pregnant women and healthy non-pregnant women and men. Graphical Abstract Temporal sensor response corresponding to respective steps of the assay for detection of PAPP-A2 in buffer.

Testing gold nanostructures fabricated by hole-mask colloidal lithography as potential substrates for SERS sensors: sensitivity, signal variability, and the aspect of adsorbate deposition.

Gold nanoplasmonic substrates with high sensitivity and spectral reproducibility are key components of molecular sensors based on surface-enhanced Raman scattering (SERS). In this work, we used a confocal Raman microscope and several types of gold nanostructures (arrays of nanodiscs, nanocones and nanodisc dimers) prepared by hole-mask colloidal lithography (HCL) to determine the sources of variability in SERS measurements. We demonstrate that significant variations in the SERS signal can originate from the method of deposition of analyte molecules onto a SERS substrate. While the method based on incubation of SERS substrates in a solution containing the analyte yields a SERS signal with low variability, the droplet deposition method produces a SERS signal with rather high variability. Variability of the SERS signal of a single nanoparticle was determined from the statistical analysis of the SERS signal in short-range Raman maps recorded using different sized laser spots produced by means of different objectives. We show that the number of nanoparticles located within the laser spot can be a source of substantial SERS signal variability, especially for high-magnification objectives. We demonstrate that SERS substrates prepared by HCL exhibit high SERS enhancement and excellent homogeneity (about 20% relative standard deviation from short-range maps). The nanocone arrays are shown to provide the highest SERS enhancement, the lowest relative level of fluorescence background, and also slightly better homogeneity when compared with arrays of nanodisc dimers or single nanodiscs.

5'-O-Methylphosphonate nucleic acids--new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes.

Several oligothymidylates containing various ratios of phosphodiester and isopolar 5'-hydroxyphosphonate, 5'-O-methylphosphonate and 3'-O-methylphosphonate internucleotide linkages were examined with respect to their hybridization properties with oligoriboadenylates and their ability to induce RNA cleavage by ribonuclease H (RNase H). The results demonstrated that the increasing number of 5'-hydroxyphosphonate or 5'-O-methylphosphonate units in antisense oligonucleotides (AOs) significantly stabilizes the heteroduplexes, whereas 3'-O-methylphosphonate AOs cause strong destabilization of the heteroduplexes. Only the heteroduplexes with 5'-O-methylphosphonate units in the antisense strand exhibited a significant increase in Escherichia coli RNase H cleavage activity by up to 3-fold (depending on the ratio of phosphodiester and phosphonate linkages) in comparison with the natural heteroduplex. A similar increase in RNase H cleavage activity was also observed for heteroduplexes composed of miRNA191 and complementary AOs containing 5'-O-methylphosphonate units. We propose for this type of AOs, working via the RNase H mechanism, the abbreviation MEPNA (MEthylPhosphonate Nucleic Acid).

Biosensor enhancement using grooved micromixers: part I, numerical studies.

In this study we examine the use of the staggered herringbone mixer (SHM) to increase the efficiency of analyte delivery to a planar biosensor surface. Although there has been an extensive amount of research regarding the optimization of the SHM for mixing purposes, there has been very little work regarding the use of said micromixers for sensing purposes. Here, we use numerical methods to examine the effect of the SHM geometry on the efficiency of analyte delivery to a biosensor surface. We show the level of sensing enhancement of an SHM-based sensing chamber over that of an unmixed chamber has a strong dependence on the SHM geometry, the Péclet number, and the overall sensor length. The results presented herein are applicable to a very wide range of biosensor transduction mechanisms and target analytes.

Biosensor Enhancement Using Grooved Micromixers: Part II, Experimental Studies.

In this study we examine the experimental use of the staggered herringbone mixer (SHM) for the signal enhancement of a microfluidic surface plasmon resonance imaging (SPRi) affinity-based biosensor. We define the signal enhancement (Emix) as the ratio of the time-dependent slope of the sensor response of a SHM-based microfluidic channel and that of an unmixed channel; Emix is directly proportional to changes in the sensor sensitivity and inversely proportional to changes in the sensor limit of detection (LOD). Measurements were carried out for three SHM designs under a wide range of volumetric flow rates for two analytes: high diffusivity ssDNA and low diffusivity Escherichia coli bacteria. The experimental data collected in this study was found to exhibit a good match to that predicted by the numerical methods discussed in part I of this study. We found that Emix is dependent on the SHM groove geometry, the Péclet number Pe, and the overall microchannel length L; these dependencies are discussed in detail. For realistic experimental conditions, the enhancement that the SHM can provide is in the range of 1 < Emix < 5 (0% < improvement < 400%).

Functionalizable low-fouling coatings for label-free biosensing in complex biological media: advances and applications.

This review focuses on recent advances in the development of functionalizable antifouling coatings and their applications in label-free optical biosensors. Approaches to the development of antifouling coatings, ranging from self-assembled monolayers and PEG derivatives to ultra-low-fouling polymer brushes, are reviewed. Methods of preparation and characterization of antifouling coatings and the functionalization of antifouling coatings with bioreceptors are reviewed, and the effect of functionalization on the fouling properties of biofunctional coating is discussed. Special attention is given to biofunctional coatings for label-free bioanalysis of blood plasma and serum for medical diagnostics.

Monitoring RAYT activity by surface plasmon resonance biosensor.

The process of DNA transposition involves the binding, cleavage, and recombination of specific DNA segments (transposable elements, TE) and is catalyzed by special enzymes encoded by the TE transposases. REP-associated tyrosine transposases (RAYTs) are a class of Y1 nucleases related to the IS200/IS605 transposases associated with a bacterial TE known as repetitive extragenic palindrome elements (REPs). Although RAYT has been subject of numerous studies, where DNA binding and cleavage by RAYT have been confirmed for Escherichia coli, the molecular mechanism of DNA insertion has not been fully understood. In this work, it is demonstrated that surface plasmon resonance (SPR) biosensor technology combined with a system of DNA hairpin probes (mimicking the natural REP sequence) and short oligonucleotides (ONs) can provide a rapid and real-time platform for monitoring and quantification of RAYT activity. We utilized RAYT from E. coli (strain MG1655) as a model system, where we evaluated its activity towards both a natural REP sequence as well as REP sequences having modifications targeting specific features of the DNA crucial for the DNA binding and cleavage. The characteristics of the RAYT-DNA interaction obtained by means of the SPR approach were compared with the results of SDS-PAGE analysis.

Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells.

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R. A collection of 34 IL-23R-binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL-23, or the biologically active human IL-23 cytokine, to the recombinant IL-23R or soluble IL-23R-IgG chimera. The strongest competitors for IL-23R binding in ELISA were confirmed to recognize human IL-23R-IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub- to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K-562, THP-1 and Jurkat, and this binding correlated with IL-23R cell-surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP-1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL-23-driven expansion of IL-17-producing primary human CD4(+) T-cells. Thus, we conclude that unique IL-23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti-inflammatory biologicals.