Functionalization of hybrid surface microparticles for in vitro cellular antigen classification.

Hybrid material surfaces on microparticles are emerging as vehicles for many biomedical multiplexing applications. Functionalization of these hybrid surface microparticles to biomolecules presents unique challenges related to optimization of surface chemistries including uniformity, repeatability, and sample sparring. Hybrid interfaces between microlevel surfaces and individual biomolecules will provide different microenvironments impacting the surface functionalization optimization and efficiency. Here, we propose and validate the first demonstration of streptavidin adsorption-based antibody functionalization on unmodified, hybrid surface microparticles for in vitro analysis. We test this analytical technique and fabricate hybrid surface microparticles with a polystyrene core and aluminum oxide semi-coating. Additionally, we optimize the streptavidin-biotin functionalization chemistry in both assay implementation and sample sparring via analytical mass balances for these microparticles and subsequently conjugate anti-human CD11b antibodies. Result confirmation and characterization occurs from ultraviolet protein absorbance and ImageJ processing of fluorescence microscopy images. Additionally, we design and implement the multi-sectional imaging (MSI) approach to support functionalization uniformity on the hybrid surface microparticles. Finally, as a proof-of-concept performance, we validate anti-CD11b antibodies functionalization by visualizing hybrid surface microparticles conjugate to human neutrophils isolated from blood samples collected from potentially septic patients. Our study introduces and defines a category of functionalization for hybrid surface microparticles with the intent of minuscule sample volumes, low cost, and low environmental impact to be used for many cellular or proteomic in vitro multiplexing applications in the future. Graphical abstract.

Plasmonic Biosensing with Aluminum Thin Films under the Kretschmann Configuration.

Aluminum has recently attracted considerable interest as a plasmonic material due to its unique optical properties, but most work has been limited to nanostructures. We report here SPR biosensing with aluminum thin-films using the standard Kretschmann configuration that has previously been dominated by gold films. Electron-beam physical vapor deposition (EBPVD)-prepared Al films oxidize in air to form a nanofilm of Al2O3, yielding robust stability for sensing applications in buffered solutions. FDTD simulations revealed a sharp plasmonic dip in the visible range that enables measurement of both angular shift and reflection intensity change at a fixed angle. Bulk and surface tests indicated that Al films exhibited superb sensitivity performance in both categories. Compared to Au, the Al/Al2O3 layer showed a marked effect of suppressing nonspecific binding from proteins in human serum. Further characterization indicated that Al film demonstrated a higher sensitivity and a wider working range than Au films when used for SPR imaging analysis. Combined with its economic and manufacturing benefits, the Al thin-film has the potential to become a highly advantageous plasmonic substrate to meet a wide range of biosensing needs in SPR configurations.

Fake news? Biotin interference in thyroid immunoassays.

We report on a 47 year old male patient with multiple sclerosis (MS) presenting in our outpatient neurology clinic in Frankfurt/Main for therapy evaluation. Before change of treatment laboratory investigations were performed. Thyroid function tests (TFTs) with a streptavidin/biotin based immunoassay revealed severe hyperthyroidism with positive thyroid autoantibodies suggestive for Graves' disease. Clinical presentation and thyroid sonography were unremarkable. Due to the discordance between clinical presentation and TFTs, we repeated medical history, in which the patient reported taking high-doses of biotin (300 mg/day) for MS. Recent studies with patients suffering from primary and secondary progressive MS, indicated promising effects of high-dose biotin on MS-related disability. In immunoassays relaying on streptavidin-biotin interaction, biotin intake can cause falsely high or low results. Two weeks after withdrawing biotin, biotin/streptavidin dependant assays showed no longer the biochemical picture of severe hyperthyroidism. Biotin intake should be paused for at least two to five days prior to the use of biotin/streptavidin dependant assays. Alternatively, non-biotin/streptavidin dependant assays (radioimmunoassay, gas chromatography-mass spectrometry/liquid chromatography-mass spectrometry) may be used.

Hybrid cavity-coupled plasmonic biosensors for low concentration, label-free and selective biomolecular detection.

Simple optical techniques that can accurately and selectively identify organic and inorganic material in a reproducible manner are of paramount importance in biological sensing applications. In this work, we demonstrate that a nanoimprinted plasmonic pattern with locked-in dimensions supports sharp deterministic hybrid resonances when coupled with an optical cavity suitable for high sensitive surface detection. The surface sensing property of this hybrid system is quantified by precise atomic layer growth of aluminum oxide using the atomic layer deposition technique. The analyte specific sensing ability is demonstrated in the detection of two dissimilar analytes, inorganic amine-coated iron oxide nanoparticles and organic streptavidin protein. Femto to nanomolar detection limits were achieved with the proposed coupled plasmonic system based on the versatile and robust soft nanoimprinting technique, which promises practical low cost biosensors.

Clickable and Antifouling Platform of Poly[(propargyl methacrylate)-ran-(2-methacryloyloxyethyl phosphorylcholine)] for Biosensing Applications.

A functional copolymer platform, namely, poly[(propargyl methacrylate)-ran-(2-methacryloyloxyethyl phosphorylcholine)] (PPgMAMPC), was synthesized by reversible addition-fragmentation chain-transfer polymerization. In principle, the alkyne moiety of propargyl methacrylate (PgMA) should serve as an active site for binding azide-containing molecules via a click reaction, i.e., Cu-catalyzed azide/alkyne cycloaddition (CuAAC), and 2-methacryloyloxyethyl phosphorylcholine (MPC), the hydrophilic monomeric unit, should enable the copolymer to suppress nonspecific adsorption. The copolymers were characterized using Fourier transform infrared (FTIR) and (1)H NMR spectroscopies. Thiol-terminated, PPgMAMPC-SH, obtained by aminolysis of PPgMAMPC, was immobilized on a gold-coated substrate using a "grafting to" approach via self-assembly. Azide-containing species, namely, biotin and peptide nucleic acid (PNA), were then immobilized on the alkyne-containing copolymeric platform via CuAAC. The potential use of surface-attached PPgMAMPC in biosensing applications was shown by detection of specific target molecules, i.e., streptavidin (SA) and DNA, by the developed sensing platform using a surface plasmon resonance technique. The copolymer composition strongly influenced the performance of the developed sensing platform in terms of signal-to-noise ratio in the case of the biotin-SA system and hybridization efficiency and mismatch discrimination for the PNA-DNA system.

Microfluidic biosensor for the detection of DNA by fluorescence enhancement and the following streptavidin detection by fluorescence quenching.

We reported an optical DNA/protein microfluidic sensor which consists of single stranded (ss) DNA-Cy3 probes on gold surface and simple line-shape microfluidic channel. These ssDNA-Cy3 probes with random sequence in bulk solution or on gold surface exhibits fluorescence enhancement after binding with complementary ssDNA (cssDNA) targets. Particularly it did not require complicated design or hairpin-like stem-loop conformation, which made it easier to be made and applied in analytes detection by fluorescence switching techniques. Using ssDNA-cy3 probes attached on gold surface in a microfluidic channel, strong fluorescence enhancement was measured by ssDNA with cssDNA binding or ssDNA with cssDNA-biotin binding. The following introduction of streptavidin resulted in fluorescence quenching (fluorescence decrease) because of the binding of hybridized DNA-biotin with streptavidin. This sensor showed strong affinity and high sensitivity toward the streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 60 amol in this microfluidic channel. Microfluidic channel height and flow rate is optimized to increase surface reaction efficiency and fluorescence switching efficiency. In contrast to previously reported optical molecular beacon approach, this sensor can be used not only for the detection of cssDNA target, but also for the detection of streptavidin. This microfluidic sensor offers the promise of analyzing kinds of molecular targets or immunoreactions.

A highly sensitive and label free biosensing platform for wireless sensor node system.

In this paper, we propose a radio-frequency (RF) biosensor platform based on oscillation frequency deviation at 2.4 GHz. Its feasibility is experimentally demonstrated with the well-known biomolecular binding systems such as biotin-streptavidin and deoxyribonucleic acid (DNA) hybridization. For a basic principle of our biosensing system, the impedance of a resonator with the biomolecular immobilization is at first varied so that the corresponding change results in frequency change of an oscillator. Especially, to enhance the sensitivity of the proposed system, a surface acoustic wave (SAW) filter having a high-Q factor (~2000) is utilized. From the resulting component, even a small change of oscillation frequency can be transformed into a large output amplitude variation. According to the experimental results, it is found that our system shows the low detectable limit (~1 ng/ml) and fast response time (~real-time) for different target biomolecules, i.e. streptavidin and complementary DNA (cDNA). As a result, we find that our device is an effective biosensing system that can be used for a label-free and real-time measurement of the biomolecular binding events.

Highly sensitive fluorescence detection of avidin/streptavidin with an optical interference mirror slide.

This paper presents highly sensitive fluorescence detections of avidin and streptavidin using an optical interference mirror (OIM) slide consisting of a plane mirror covered with an optical interference layer. Compared with a common glass slide, the OIM slide can enhance the fluorescence from a dye by more than 100-fold. We fabricated an OIM slide by depositing an optical interference layer of Al(2)O(3) on an Ag mirror. To enhance the fluorescence maximally, the optimal thickness of the Al(2)O(3) layer was estimated from optical interference theory. For detections of protein, avidin/streptavidin labeled with fluorescein, Cy3, and Cy5 were detected with biotin immobilized on an OIM slide with the optimal Al(2)O(3) thickness. We achieved a sensitivity improvement of more than 50-fold, comparing with a glass slide. Such a high degree of improvement would be a significant contribution to further progress in biomedical research and medical diagnostics.

Biomolecular interaction analysis under electrophoretic flow conditions.

Combining the advantages of electrophoresis with the advantages of biomolecular interaction analysis (BIA) enables the biospecific detection of separated molecules; for example it permits differentiation between a complementary single-stranded DNA and a single nucleotide polymorphism. In order to integrate these two techniques, it is necessary to investigate whether it is possible to detect a biomolecular interaction under electrophoretic flow conditions. To this end a novel detection system was developed for electrophoresis that utilizes a label-free and time-resolved detection technique: reflectometric interference spectroscopy (RIfS). The biological functions of important analytes were investigated using this system. Although RIfS can be used as a postcolumn detector, it is also possible to use it to detect relevant substances under electrophoretic flow conditions. DNA-LNA, biotin-streptavidin and protein-protein interactions were detected using this coupled electrophoresis-RIfS set-up.

The solution to the streptavidin-biotin paradox: the influence of history on the strength of single molecular bonds.

In the past few years, many studies have attempted to measure the strength of a single molecular bond. In general, these experiments consisted in pulling on the bond and measuring the force necessary to dissociate the molecules. However, seemingly contradictory experimental results led to draw the intriguing conclusion that the strength of the bond could depend on the experiment even if the pulling conditions are similar: this paradox was first observed on the widely used streptavidin-biotin bond. Here, by doing supplementary measurements and by reanalyzing the controversial experimental results using Kramers' theory, we show that they can be conciliated. This allows us to show that the strength of a bond is very sensitive to the history of its formation, which is the key to the paradox.

Characterization of DNA immobilization and subsequent hybridization on a 2D arrangement of streptavidin on a biotin-modified lipid bilayer supported on SiO2.

We show how the water content (and effective density) of thin adsorbed films composed of biomolecules can be determined using combined quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) analysis. In particular, these techniques, combined with theoretical treatment using a Voigt-based viscoelastic model, were applied to analyze the state of surface immobilized single stranded biotin-modified probe DNA (b-DNA) coupled via streptavidin to a biotin-doped supported phospholipid bilayer (b-SPB)). From a proper analysis, it is demonstrated how changes in effective thickness, delta(f), and the viscoelastic components (shear viscosity, eta(f), and shear elasticity, mu(f))) can be obtained during both DNA immobilization and hybridization with single stranded fully complementary target DNA. In particular, it is demonstrated how this type of analysis can be used to control the state of streptavidin arrangement for improved measurements of DNA hybridization kinetics. The latter is demonstrated by identifying a surface-coverage dependent viscoelastic behavior of immobilized b-DNA, which is shown to influence the hybridization efficiency.