Superior LSPR substrates based on electromagnetic decoupling for on-a-chip high-throughput label-free biosensing.

Localized surface plasmon resonance (LSPR) biosensing based on supported metal nanoparticles offers unparalleled possibilities for high-end miniaturization, multiplexing and high-throughput label-free molecular interaction analysis in real time when integrated within an opto-fluidic environment. However, such LSPR-sensing devices typically contain extremely large regions of dielectric materials that are open to molecular adsorption, which must be carefully blocked to avoid compromising the device readings. To address this issue, we made the support essentially invisible to the LSPR by carefully removing the dielectric material overlapping with the localized plasmonic fields through optimized wet-etching. The resulting LSPR substrate, which consists of gold nanodisks centered on narrow SiO2 pillars, exhibits markedly reduced vulnerability to nonspecific substrate adsorption, thus allowing, in an ideal case, the implementation of thicker and more efficient passivation layers. We demonstrate that this approach is effective and fully compatible with state-of-the-art multiplexed real-time biosensing technology and thus represents the ideal substrate design for high-throughput label-free biosensing systems with minimal sample consumption.

Drop coating deposition of a liposome suspension on surfaces with different wettabilities: "coffee ring" formation and suspension preconcentration.

Evaporation of a drop of biomolecular solution on a solid surface typically creates a ring-shaped drying pattern, formed by the so-called "coffee ring" effect. The size and shape of the "coffee ring" pattern is strongly dependent on the properties of the surface as well as on the deposited molecular solution or suspension. In this paper, we tested six types of surfaces differing in their physico-chemical surface characteristics (contact angles, wettability and roughness) as well as in the presence or absence of a base metal layer. The tested surfaces include two fluorocarbon coated metallic surfaces (commercial SpectRIM™ from Tienta Sciences, Inc. based on a smoothed stainless steel and non-commercial aluminium surface), three silanized glass surfaces and polished CaF2. The results showed that the formation of a "coffee ring" was influenced by surface wettability as well as by lipid concentration in the drop. Drop coating deposition Raman (DCDR) spectroscopy was used to compare the ability of the tested surfaces to preconcentrate molecules in the ring and therefore improve detection sensitivity. It was shown that surfaces with a contact angle of 90° and higher produce smaller drying patterns than more hydrophilic surfaces. In these drying patterns, the model liposomes were more efficiently preconcentrated, which resulted in a higher Raman signal of the liposomes. The applicability of surfaces with static contact angles less than 90°, high water contact angle hysteresis and no metal layer (silanized glass, CaF2) is limited to samples with high liposome concentrations.

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).

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.

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.

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).

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.

Configuration-controlled Au nanocluster arrays on inverse micelle nano-patterns: versatile platforms for SERS and SPR sensors.

Nanopatterned 2-dimensional Au nanocluster arrays with controlled configuration are fabricated onto reconstructed nanoporous poly(styrene-block-vinylpyridine) inverse micelle monolayer films. Near-field coupling of localized surface plasmons is studied and compared for disordered and ordered core-centered Au NC arrays. Differences in evolution of the absorption band and field enhancement upon Au nanoparticle adsorption are shown. The experimental results are found to be in good agreement with theoretical studies based on the finite-difference time-domain method and rigorous coupled-wave analysis. The realized Au nanopatterns are exploited as substrates for surface-enhanced Raman scattering and integrated into Kretschmann-type SPR sensors, based on which unprecedented SPR-coupling-type sensors are demonstrated.

Surface plasmon resonance sensing of nucleic acids: a review.

Biosensors based on surface plasmon resonance (SPR) have become a central tool for the investigation and quantification of biomolecules and their interactions. Nucleic acids (NAs) play a vital role in numerous biological processes and therefore have been one of the major groups of biomolecules targeted by the SPR biosensors. This paper discusses the advances of NA SPR biosensor technology and reviews its applications both in the research of molecular interactions involving NAs (NA-NA, NA-protein, NA-small molecule), as well as for the field of bioanalytics in the areas of food safety, medical diagnosis and environmental monitoring.

Enhancement of affinity-based biosensors: effect of sensing chamber geometry on sensitivity.

Affinity-based biosensing systems have become an important analytical tool for the detection and study of numerous biomolecules. The merging of these sensing technologies with microfluidic flow cells allows for faster detection times, increased sensitivities, and lower required sample volumes. In order to obtain a higher degree of performance from the sensor, it is important to know the effects of the flow cell geometry on the sensor sensitivity. In these sensors, the sensor sensitivity is related to the overall diffusive flux of analyte to the sensing surface; therefore increases in the analyte flux will be manifested as an increase in sensitivity, resulting in a lower limit of detection (LOD). Here we present a study pertaining to the effects of the flow cell height H on the analyte flux J, where for a common biosensor design we predict that the analyte flux will scale as J ≈ H(-2/3). We verify this scaling behavior via both numerical simulations as well as an experimental surface plasmon resonance (SPR) biosensor. We show the reduction of the flow cell height can have drastic effects on the sensor performance, where the LOD of our experimental system concerning the detection of ssDNA decreases by a factor of 4 when H is reduced from 47 µm to 7 µm. We utilize these results to discuss the applicability of this scaling behavior with respect to a generalized affinity-based biosensor.

Ultrasensitive broadband probing of molecular vibrational modes with multifrequency optical antennas.

Optical antennas represent an enabling technology for enhancing the detection of molecular vibrational signatures at low concentrations and probing the chemical composition of a sample in order to identify target molecules. However, efficiently detecting different vibrational modes to determine the presence (or the absence) of a molecular species requires a multispectral interrogation in a window of several micrometers, as many molecules present informative fingerprint spectra in the mid-infrared between 2.5 and 10 µm. As most nanoantennas exhibit a narrow-band response because of their dipolar nature, they are not suitable for such applications. Here, we propose the use of multifrequency optical antennas designed for operating with a bandwidth of several octaves. We demonstrate that surface-enhanced infrared absorption gains in the order of 10(5) can be easily obtained in a spectral window of 3 µm with attomolar concentrations of molecules, providing new opportunities for ultrasensitive broadband detection of molecular species via vibrational spectroscopy techniques.

High-resolution biosensor based on localized surface plasmons.

We report on a new biosensor with localized surface plasmons (LSP) based on an array of gold nanorods and the total internal reflection imaging in polarization contrast. The sensitivity of the new biosensor is characterized and a model detection of DNA hybridization is carried out. The results are compared with a reference experiment using a conventional high-resolution surface plasmon resonance (SPR) biosensor. We show that the LSP-based biosensor delivers the same performance as the SPR system while involving significantly lower surface densities of interacting molecules. We demonstrate a limit of detection of 100 pM and a surface density resolution of only 35 fg×mm-2 that corresponds to less than one DNA molecule per nanoparticle on average.

Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G.

Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high-affinity binders of human interferon gamma (hIFNγ) from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin-binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNγ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited K(d) values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ, and in vitro competition for hIFNγ binding revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNγ, which differed from the site of human IFNγ receptor 1 binding. Thus, these hIFNγ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold.

Analytical value of detecting an individual molecular binding event: the case of the surface plasmon resonance biosensor.

We explore the ultimate limits of the performance of bioanalytical approaches based on the detection of individual molecular binding events taking place at the sensor surface interfaced with a microfluidic flow-through cell. As a case study, we investigate and compare the bioanalytical performance of flow-through surface plasmon resonance (SPR) sensors based on (1) localized surface plasmons (LSP) which detect a single binding event and (2) propagating surface plasmons (PSP) which integrate a great number of simultaneously occurring binding events. We demonstrate that for the biomolecular interactions most relevant to biosensing the single-binding-event LSP approach is inferior to the integrating PSP approach. We estimate that the number of biorecognition elements available to interact with the analyte molecules would need to be, depending on the size of the analyte and parameters of the molecular interaction, in the order of 10 to 10(3) to increase the probability of the positive response of the LSP-based sensor to that of the PSP-based sensor.

Streptavidin-enhanced assay for sensitive and specific detection of single nucleotide polymorphism in TP53.

This paper reports an approach to detection of single nucleotide polymorphism based on special amplification assay and surface plasmon resonance biosensor technology. In this assay, a part of the target DNA is recognized by a probe (probe A) coupled with streptavidin-oligonucleotide (SON) complexes ex situ, and when the mixture is injected in the sensor, another part of the target DNA is recognized by a DNA probe (probe B) immobilized on the sensor surface. To achieve high sensitivity and specificity, the assay is optimized in terms of composition of SON complexes, probe design, and assay temperature. It is demonstrated that this approach provides high specificity (no response to targets containing single-mismatched bases) and sensitivity (improves sensor response to perfectly matched oligonucleotides by one order of magnitude compared to the direct detection method). The assay is applied to detection of a short synthetic analogue of TP53 containing a "hot spot"-single nucleotide mismatch frequently mutated in germ line cancer-at levels down to 40 pM.

Surface plasmon resonance biosensor for rapid label-free detection of microribonucleic acid at subfemtomole level.

Microribonucleic acids (miRNAs) have been linked with various regulatory functions and disorders, such as cancers and heart diseases. They, therefore, present an important target for detection technologies for future medical diagnostics. We report here a novel method for rapid and sensitive miRNA detection and quantitation using surface plasmon resonance (SPR) sensor technology and a DNA*RNA antibody-based assay. The approach takes advantage of a novel high-performance portable SPR sensor instrument for spectroscopy of surface plasmons based on a special diffraction grating called a surface plasmon coupler and disperser (SPRCD). The surface of the grating is functionalized with thiolated DNA oligonucleotides which specifically capture miRNA from a liquid sample without amplification. Subsequently, an antibody that recognizes DNA*RNA hybrids is introduced to bind to the DNA*RNA complex and enhance sensor response to the captured miRNA. This approach allows detection of miRNA in less than 30 min at concentrations down to 2 pM with an absolute amount at high attomoles. The methodology is evaluated for analysis of miRNA from mouse liver tissues and is found to yield results which agree well with those provided by the quantitative polymerase chain reaction (qPCR).

A dual surface plasmon resonance assay for the determination of ribonuclease H activity.

There is a demand for efficient tools for the monitoring of RNase H activity. We report on a new assay which allows for simultaneous (1) real-time monitoring of RNase H activity and (2) detection of cleavage reaction products. The dual assay is implemented using a multichannel surface plasmon resonance (SPR) biosensor with two independently functionalized sensing areas in a single fluidic path. In the first sensing area the RNA cleavage by RNase H is monitored, while the products of the cleavage reaction are captured in the second sensing area with specific DNA probes. The assay was optimized with respect to AON concentration and temperature. A significant improvement was obtained with special chimeric probes, which contain RNA substrate for RNase H and a longer deoxyribonucleotide tail, which enhances the SPR signal. It has been shown that RNase H stabilizes the RNA:DNA hybrid duplex before the cleavage. The potential of the assay is demonstrated in the study in which the ability of natural and modified oligonucleotides to activate RNase H is examined.

Shielding effect of monovalent and divalent cations on solid-phase DNA hybridization: surface plasmon resonance biosensor study.

Solid-phase hybridization, i.e. the process of recognition between DNA probes immobilized on a solid surface and complementary targets in a solution is a central process in DNA microarray and biosensor technologies. In this work, we investigate the simultaneous effect of monovalent and divalent cations on the hybridization of fully complementary or partly mismatched DNA targets to DNA probes immobilized on the surface of a surface plasmon resonance sensor. Our results demonstrate that the hybridization process is substantially influenced by the cation shielding effect and that this effect differs substantially for solid-phase hybridization, due to the high surface density of negatively charged probes, and hybridization in a solution. In our study divalent magnesium is found to be much more efficient in duplex stabilization than monovalent sodium (15 mM Mg2+ in buffer led to significantly higher hybridization than even 1 M Na+). This trend is opposite to that established for oligonucleotides in a solution. It is also shown that solid-phase duplex destabilization substantially increases with the length of the involved oligonucleotides. Moreover, it is demonstrated that the use of a buffer with the appropriate cation composition can improve the discrimination of complementary and point mismatched DNA targets.

Surface plasmon resonance study on HIV-1 integrase strand transfer activity.

Understanding the molecular mechanism of HIV-1 integrase (IN) activity is critical to find functional inhibitors for an effective AIDS therapy. A robust, fast, and sensitive method for studying IN activity is required. In this work, an assay for real-time label-free monitoring of the IN activity based on surface plasmon resonance was developed. This assay enabled direct monitoring of the integration of a viral doubled-stranded (ds) DNA into the host genome. The strand transfer reaction was detected by using two different DNA targets: supercoiled plasmid (pUC 19) and short palindrome oligonucleotide. The effect of the length of the DNA target on the possibility to monitor the actual process of the strand transfer reaction is discussed. The surface density of integrated ds-DNA was determined. IN binding to the oligonucleotide complexes and model DNA triplexes in the presence of various divalent ions as metal cofactors was investigated as well. The assay developed can serve as an important analytical tool to search for potential strand transfer reaction inhibitors as well as for the study of compounds interfering with the binding of ds long terminal repeats-IN complexes with the host DNA.