Markéta Bocková

Surface plasmon resonance biosensor for parallelized detection of protein biomarkers in diluted blood plasma

Surface plasmon resonance (SPR) biosensor for high-throughput screening of protein biomarkers in diluted blood plasma is reported. The biosensor combines a high-resolution SPR imaging sensor and a high-density protein array with low-fouling background. The SPR imaging sensor utilizes polarization contrast and advanced referencing and provides a total of 120 sensing areas (each 200 μm×150 μm). Antibodies are immobilized on the sensing areas via hybridization of antibody-oligonucleotide conjugates to thiolated complementary oligonucleotides microspotted on the sensor surface (DNA-directed immobilization). A low-fouling background is achieved by covalent immobilization of bovine serum albumin to carboxyl-terminated thiols filling the areas among the thiolated oligonucleotides and outside the sensing areas. The biosensor was evaluated for detection of protein biomarkers relevant to cancer diagnostics--human chorionic gonadotropin (hCG) and activated leukocyte cell adhesion molecule (ALCAM) both in buffer and in 10% blood plasma. Limits of detection as low as 45 ng/mL (ALCAM) and 100 ng/mL (hCG) were achieved in blood plasma samples.

Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor

We report an ultra-low fouling surface plasmon resonance imaging (SPRi) biosensor for the rapid simultaneous detection of multiple miRNAs in erythrocyte lysate (EL) at subpicomolar levels without need of RNA extraction. The SPRi chips were coated with ultra-low fouling functionalizable poly(carboxybetaine acrylamide) (pCBAA) brushes having optimized thicknesses and directly functionalized with amino-modified oligonucleotide probes. We have characterized the effect of the brush thickness on the probe loading capacity: a loading capacity of ~9.8×10(12) probes/cm(2) was achieved for pCBAA having a thickness of ~40 nm. The probe-functionalized sensor also exhibited a high resistance to fouling from ~90% EL samples (<2 ng/cm(2)). A two-step detection assay was employed for multiplexed miRNA detection in EL. Specifically, the assay consisted of (i) a sandwich-type hybridization of the probe-functionalized pCBAA with target miRNA in EL (bound to biotinylated oligonucleotides) and (ii) the capture of streptavidin-functionalized gold nanoparticles to the aforementioned biotinylated probes. We have demonstrated that this approach enables the detection of miRNAs in EL at concentrations as low as 0.5 pM. Finally, we have confirmed the detection of four endogenous miRNAs representing a set of potential miRNA biomarkers of myelodysplastic syndrome (MDS) in clinical EL samples (miR-16, miR-181, miR-34a, and miR-125b). The results revealed significantly higher levels of miR-16 in all the clinical EL samples compared to the other measured miRNAs.

Label-Free Biosensing in Complex Media: A Referencing Approach

We present a novel approach to reference-compensated label-free affinity biosensing in complex media. Unlike conventional approaches that employ surfaces with different biological functionalities in the detection and reference channels to produce a reference-compensated sensor response, the new approach (referred as to single surface referencing (SSR)) uses a single functionalized surface split into the detection and reference channel to which complex sample (detection channel) and complex sample mixed with biomolecules binding to the analyte and thus inhibiting the binding of the analyte to the functionalized surface (reference channel) is introduced. This approach ensures that (i) only the detection channel captures the analyte and (ii) nonspecific binding incurred in the detection and reference channels are the same. We evaluate this approach in a model biosensing experiment, detection of a cancer biomarker carcinoembryonic antigen (CEA) in blood plasma using antibody against CEA and a surface plasmon resonance (SPR) biosensor. We detect CEA in three different blood plasma samples and demonstrate that this novel referencing approach provides more accurate results and lower biological variability than the conventional referencing.

Enhanced levels of mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 in patients with Alzheimer disease and multiple sclerosis

The multifunctional mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 might play a role in the development of Alzheimer disease via its high-affinity binding to amyloid beta peptides and its neuronal over-expression. It is suggested that the cerebrospinal fluid levels of the enzyme, free or bound to amyloid beta peptides, are a potential specific biomarker of Alzheimer disease. However, mitochondrial dysfunction seems to play a role in many neurological diseases including multiple sclerosis. In this study, the specificity of changes in relation to the enzyme over-expression was evaluated using enzyme-linked immunosorbent and surface plasmon resonance sensors. The data indicated pronounced increases in the enzyme levels, specifically to 179% in multiple sclerosis and to 573% in Alzheimer disease when compared to the age-matched controls. Although the differences between both diseases were statistically significant, enzyme levels do not appear to be a highly specific biomarker of Alzheimer disease. On the other hand, enhancement in levels of the enzyme bound to amyloid beta peptides was only observed in people with Alzheimer disease, which suggests that the complex should be further considered as a possible biomarker. In patients with multiple sclerosis, our results are the first to demonstrate significant changes in enzyme expression and to suggest possible alterations in amyloid beta peptides.

Real-time label-free monitoring of the cellular response to osmotic stress using conventional and long-range surface plasmons.

Cell volume and its regulation are key factors for cellular integrity and also serve as indicators of various cell pathologies. SPR sensors represent an efficient tool for real-time and label-free observations of changes in cell volume and shape. Here, we extend this concept by employing the use of long-range surface plasmons (LRSP). Due to the enhanced penetration depth of LRSP (~1μm, compared to ~0.4μm of a conventional surface plasmon), the observation of refractive index changes occurring deeper inside the cells is possible. In this work, the responses of a confluent normal rat kidney (NRK) epithelial cell layer to osmotic stress are studied by both conventional and long-range surface plasmons. Experiments are conducted in parallel using cell layers grown and stimulated under the same conditions to enable direct comparison of the results and discrimination of the osmotic stress-induced effects in different parts of the cell.

Toward single-molecule detection with sensors based on propagating surface plasmons

Surface plasmon resonance (SPR) sensors are known to be able to detect very low surface concentrations of (bio)molecules on macroscopic areas. To explore the potential of SPR biosensors to achieve single-molecule detection, we have minimized the read-out area (to ~64 μm2) by employing a sensor system based on spectroscopy of surface plasmons generated on a diffractive structure via a microscope objective and light collection through a small aperture. This approach allows for decreasing the number of detected molecules by 3 orders of magnitude compared to state-of-the-art SPR sensors. A protein monolayer has been shown to produce a response of 5000 times the baseline noise, suggesting that as few as ~500 proteins could be detected by the sensor.

Biosensing enhancement using passive mixing structures for microarray-based sensors.

The combination of microarray technologies with microfluidic sample delivery and real-time detection methods has the capability to simultaneously monitor 10-1000 s of biomolecular interactions in a single experiment. Despite the benefits that microfluidic systems provide, they typically operate in the laminar flow regime under mass transfer limitations, where large analyte depletion layers act as a resistance to analyte capture. By locally stirring the fluid and delivering fresh analyte to the capture spot, the use of passive mixing structures in a microarray environment can reduce the negative effects of these depletion layers and enhance the sensor performance. Despite their large potential, little attention has been given to the integration of these mixing structures in microarray sensing environments. In this study, we use passive mixing structures to enhance the mass transfer of analyte to a capture spot within a microfluidic flow cell. Using numerical methods, different structure shapes and heights were evaluated as means to increase local fluid velocities, and in turn, rates of mass transfer to a capture spot. These results were verified experimentally via the real-time detection of 20-mer ssDNA for an array of microspots. Both numerical and experimental results showed that a passive mixing structure situated directly over the capture spot can significantly enhance the binding rate of analyte to the sensing surface. Moreover, we show that these structures can be used to enhance mass transfer in experiments regarding an array of capture spots. The results of this study can be applied to any experimental system using microfluidic sample delivery methods for microarray detection techniques.

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%).

The Scavenger Receptor SSc5D Physically Interacts with Bacteria through the SRCR-Containing N-Terminal Domain

The scavenger receptor cysteine-rich (SRCR) family comprises a group of membrane-attached or secreted proteins that contain one or more modules/domains structurally similar to the membrane distal domain of type I macrophage scavenger receptor. Although no all-inclusive biological function has been ascribed to the SRCR family, some of these receptors have been shown to recognize pathogen-associated molecular patterns (PAMP) of bacteria, fungi, or other microbes. SSc5D is a recently described soluble SRCR receptor produced by monocytes/macrophages and T lymphocytes, consisting of an N-terminal portion, which contains five SRCR modules, and a large C-terminal mucin-like domain. Toward establishing a global common role for SRCR domains, we interrogated whether the set of five SRCR domains of SSc5D displayed pattern recognition receptor (PRR) properties. For that purpose, we have expressed in a mammalian expression system the N-terminal SRCR-containing moiety of SSc5D (N-SSc5D), thus excluding the mucin-like domain likely by nature to bind microorganisms, and tested the capacity of the SRCR functional groups to physically interact with bacteria. Using conventional protein–bacteria binding assays, we showed that N-SSc5D had a superior capacity to bind to Escherichia coli strains RS218 and IHE3034 compared with that of the extracellular domains of the SRCR proteins CD5 and CD6 (sCD5 and sCD6, respectively), and similar E. coli-binding properties as Spα, a proven PRR of the SRCR family. We have further designed a more sensitive, real-time, and label-free surface plasmon resonance (SPR)-based assay and examined the capacity of N-SSc5D, Spα, sCD5, and sCD6 to bind to different bacteria. We demonstrated that N-SSc5D compares with Spα in the capacity to bind to E. coli and Listeria monocytogenes, and further that it can distinguish between pathogenic E. coli RS218 and IHE3034 strains and the non-pathogenic laboratory E. coli strain BL21(DE3). Our work thus advocates the utility of SPR-based assays as sensitive tools for the rapid screening of interactions between immune-related receptors and PAMP-bearing microbes. The analysis of our results suggests that SRCR domains of different members of the family have a differential capacity to interact with bacteria, and further that the same receptor can discriminate between different bacteria strains and species.

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.