Low cost and massively parallel force spectroscopy with fluid loading on a chip.

Current approaches for single molecule force spectroscopy are typically constrained by low throughput and high instrumentation cost. Herein, a low-cost, high throughput technique is demonstrated using microfluidics for multiplexed mechanical manipulation of up to ~4000 individual molecules via molecular fluid loading on-a-chip (FLO-Chip). The FLO-Chip consists of serially connected microchannels with varying width, allowing for simultaneous testing at multiple loading rates. Molecular force measurements are demonstrated by dissociating Biotin-Streptavidin and Digoxigenin-AntiDigoxigenin interactions along with unzipping of double stranded DNA of varying sequence under different dynamic loading rates and solution conditions. Rupture force results under varying loading rates and solution conditions are in good agreement with prior studies, verifying a versatile approach for single molecule biophysics and molecular mechanobiology. FLO-Chip enables straightforward, rapid, low-cost, and portable mechanical testing of single molecules that can be implemented on a wide range of microscopes to broaden access and may enable new applications of molecular force spectroscopy.

An Economical High-Throughput "FP-Tag" Assay for Screening Glycosyltransferase Inhibitors*.

O-GlcNAc transferase (OGT) is involved in many cellular processes, and selective OGT inhibitors are valuable tools to investigate O-GlcNAcylation functions, and could potentially lead to therapeutics. However, high-throughput OGT assays that are suitable for large-scale HTS and can identify inhibitors targeting both acceptor, donor sites, and allosteric binding-sites are still lacking. Here, we report the development of a high-throughput "FP-Tag" OGT assay with bovine serum albumin (BSA) as a low-cost and superior "FP-Tag". With this assay, 2-methyleurotinone was identified as a low-micromolar OGT inhibitor. This type of assay with BSA as "FP-Tag" would find more applications with other glycosyltransferases.

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.

Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time- and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

Programmable Multivalent DNA-Origami Tension Probes for Reporting Cellular Traction Forces.

Mechanical forces are central to most, if not all, biological processes, including cell development, immune recognition, and metastasis. Because the cellular machinery mediating mechano-sensing and force generation is dependent on the nanoscale organization and geometry of protein assemblies, a current need in the field is the development of force-sensing probes that can be customized at the nanometer-length scale. In this work, we describe a DNA origami tension sensor that maps the piconewton (pN) forces generated by living cells. As a proof-of-concept, we engineered a novel library of six-helix-bundle DNA-origami tension probes (DOTPs) with a tailorable number of tension-reporting hairpins (each with their own tunable tension response threshold) and a tunable number of cell-receptor ligands. We used single-molecule force spectroscopy to determine the probes' tension response thresholds and used computational modeling to show that hairpin unfolding is semi-cooperative and orientation-dependent. Finally, we use our DOTP library to map the forces applied by human blood platelets during initial adhesion and activation. We find that the total tension signal exhibited by platelets on DOTP-functionalized surfaces increases with the number of ligands per DOTP, likely due to increased total ligand density, and decreases exponentially with the DOTP's force-response threshold. This work opens the door to applications for understanding and regulating biophysical processes involving cooperativity and multivalency.

Bifunctional linker-based immunosensing for rapid and visible detection of bacteria in real matrices.

Detection of pathogens present in food and water is essential to help ensure food safety. Among the popular methods for pathogen detection are those based on culture and colony-counting and polymerase chain reaction (PCR). However, the time-consuming nature and/or the need for sophisticated instrumentation of those methods limit their on-site applications. We have developed a rapid and highly sensitive immunosensing method for visible detection of bacteria in real matrices based on the aggregation of AuNPs without requiring any readout device. We use biotinylated anti-bacteria antibodies as bifunctional linkers (BLs) to mediate the aggregation of streptavidin-functionalized gold nanoparticles (st-AuNPs) to produce visually recognizable color change, due to surface plasmon resonance (SPR), which occurs in about 30min of total assay time when the sample is mildly agitated or within three hours in quiescent conditions. The aggregation of st-AuNPs, which produces the indication signal, is achieved very differently than in visual detection methods reported previously and hence affords ultrahigh sensitivity. While BLs can both bind to the target and crosslink st-AuNPs, their latter function is essentially disabled when they bind to the target bacteria. By varying the amount of st-AuNPs used, we can tailor the assay effectiveness improving limit of detection (LOD) down to 10CFUmL-1 of E. coli and Salmonella. Test results obtained with tap water, lake water and milk samples show that assay performance is unaffected by matrix effects. Further, in a mixture of live and autoclaved E. coli cells our assay could detect only live cells. Therefore, our BL-based immunosensor is suitable for highly sensitive, rapid, and on-site detection of bacteria in real matrices.

Enzyme-Responsive LipoCEST Agents: Assessment of MMP-2 Activity by Measuring the Intra-liposomal Water 1 H†NMR Shift.

Mobile proton-containing solutes can be detected by MRI by the chemical exchange saturation transfer (CEST) method. CEST sensitivity is dramatically enhanced by using, as exchanging protons, the water molecules confined inside liposomes, shifted by a paramagnetic shift reagent. The chemical shift of the intraliposomal water resonance (δIL ) is affected by the overall shape of the supramolecular system. δIL of a spherical LipoCEST acts as a sensitive reporter of the distribution of streptavidin proteins anchored at the liposome surface by biotinylated phospholipids. This finding prompted the design of a MMP-2 responsive LipoCEST agent as the streptavidin moieties can be released from the liposome surfaces when a properly tailored enzyme-cleavable peptide is inserted on the phospholipids before the terminal biotin residues. δIL reports on the overall changes in the supramolecular architecture associated to the cleavage carried out by MMP-2.

Single-molecule FRET-Rosetta reveals RNA structural rearrangements during human telomerase catalysis.

Maintenance of telomeres by telomerase permits continuous proliferation of rapidly dividing cells, including the majority of human cancers. Despite its direct biomedical significance, the architecture of the human telomerase complex remains unknown. Generating homogeneous telomerase samples has presented a significant barrier to developing improved structural models. Here we pair single-molecule Förster resonance energy transfer (smFRET) measurements with Rosetta modeling to map the conformations of the essential telomerase RNA core domain within the active ribonucleoprotein. FRET-guided modeling places the essential pseudoknot fold distal to the active site on a protein surface comprising the C-terminal element, a domain that shares structural homology with canonical polymerase thumb domains. An independently solved medium-resolution structure of Tetrahymena telomerase provides a blind test of our modeling methodology and sheds light on the structural homology of this domain across diverse organisms. Our smFRET-Rosetta models reveal nanometer-scale rearrangements within the RNA core domain during catalysis. Taken together, our FRET data and pseudoatomic molecular models permit us to propose a possible mechanism for how RNA core domain rearrangement is coupled to template hybrid elongation.

Conformation-sensitive antibody-based point-of-care immunosensor for serum Ca(2+) using two-dimensional sequential binding reactions.

To assess the homeostasis of Ca(2+) metabolism, we have developed a rapid immunosensor for ionic calcium using a membrane chromatographic technique. As calcium-binding protein (CBP) is available for the recognition and undergone conformation change upon Ca(2+) binding, a monoclonal antibody sensitive to the altered structure of CBP has been employed. The sequential binding scheme was mathematically simulated and shown to match with the experimental results. At the initial stage, the rapid analytical system using lateral flow was constructed by immobilizing the antibody on the immuno-strip nitrocellulose membrane and labeling CBP with colloidal gold as a tracer. A major problem with this system in measuring ionic calcium levels was retarded migration of the gold tracer along the immuno-strip. It was conceivable that the divalent cation at a high concentration caused a change in the physical properties of the tracer, resulting in a non-specific interaction with the membrane surface. This problem was circumvented by first eluting a sample containing biotinylated CBP along the immuno-strip and then supplying the gold coupled to streptavidin across the signal generation pad of the strip. The color signal was then generated via biotin-SA linkage and measured using a smartphone-based detector developed in our laboratory. This two-dimensional chromatographic format completed the Ca(2+) analysis within 15min, the analytical performance covered the clinical dynamic range (0.25-2.5mM) and highly correlated with that of the reference system, i-STAT. These results inspired us to eventually investigate a dual-immunoassay system that measures simultaneously ionic calcium and parathyroid hormone, which regulates the ionic calcium level in serum. This will significantly simplify the current diagnostic protocols, which involve separate devices.

Assembly and Assessment of DNA Scaffolded Vaccines.

Vaccines play an important role in preventing many life-threatening infectious diseases. To meet the demand of vaccination for treating a wide range of diseases, rational vaccine design has been recognized as a desirable and necessary strategy for development of safe and effective vaccines. DNA nanostructures are advantageous in the design and construction of synthetic vaccines, owing to their robust self-assembly, programmability, and precision control in complex organization, as well as their intrinsic adjuvant activity. Here, we describe a modular assembly of DNA scaffolded vaccine complex, composing of a model antigen, streptavidin, and adjuvant, CpG oligonucleotide. The DNA-assembled vaccines were found to elicit strong antigen-specific antibody responses, but causing little or no adverse reactions. Conceivably, this vaccine platform can be further optimized for improved immunogenicity and extended to the construction of various subunit vaccines.

Linear quantification of a streptavidin-alkaline phosphatase probe for enzyme-linked immuno mass spectrometric assay.

The alkaline phosphatase-streptavidin (AP-SA) probe released adenosine (∼267.2 Da) from the substrate adenosine monophosphate (AMP), where a signal may be detected from as little as 0.5 µl of a 0.1-pg/ml dilution of the probe (2.6 × 10(-22) mol). The signal from the AP-SA probe was linear from 1 to 50 pg/ml by monitoring adenosine release at 268 m/z (M + H) with liquid chromatography, electrospray ionization, and quadrupole mass spectrometry (LC-ESI-MS). The safe limit of detection and quantification of the AP-SA probe was approximately 0.5 pg/well or 5 pg/ml. Enzyme-linked immuno mass spectrometric assay (ELIMSA) using the AP-SA probe provided a linear signal response for prostate-specific antigen (PSA) against external standards from 1 to 500 pg/ml. The ELIMSA showed a safe limit of detection and quantification at 5 pg PSA/well or 50 pg/ml (false positive detection rate P ≤ 0.01). Female samples of 100 µl plasma/well were read against standards and blanks made in normal female plasma, and the lowest sample quantified was approximately 9.8 pg/well or 98 pg/ml. Here ELIMSA was applied to measure PSA in plasma from female, normal male, prostatectomy patient, and cancer patient samples that showed significant differences by analysis of variance (ANOVA).

Improved DNA microarray detection sensitivity through immobilization of preformed in solution streptavidin/biotinylated oligonucleotide conjugates.

A novel immobilization approach involving binding of preformed streptavidin/biotinylated oligonucleotide conjugates onto surfaces coated with biotinylated bovine serum albumin is presented. Microarrays prepared according to the proposed method were compared, in terms of detection sensitivity and specificity, with other immobilization schemes employing coupling of biotinylated oligonucleotides onto directly adsorbed surface streptavidin, or sequential coupling of streptavidin and biotinylated oligonucleotides onto a layer of adsorbed biotinylated bovine serum albumin. A comparison was performed employing biotinylated oligonucleotides corresponding to wild- and mutant-type sequences of seven single point mutations of the BRCA1 gene. With respect to the other immobilization protocols, the proposed oligonucleotide immobilization approach offered the highest hybridization signals (at least 5 times higher) and permitted more elaborative washings, thus providing considerably higher discrimination between complimentary and non-complementary DNA sequences for all mutations tested. In addition, the hybridization kinetics were significantly enhanced compared to two other immobilization protocols, permitting PCR sample analysis in less than 40 min. Thus, the proposed oligonucleotide immobilization approach offered improved detection sensitivity and discrimination ability along with considerably reduced analysis time, and it is expected to find wide application in DNA mutation detection.

Flow-through, viral co-infection assay for resource-limited settings.

Here we present a new and rapid immunofiltration assay for simultaneous detection of HIV p24 and hepatitis B virus antigens. The assay platform is composed of a 13 mm nitrocellulose filter spotted with capturing bioprobes and inserted in a Swinnex(®) syringe filter holder. Samples and reagents are flown through the nitrocellulose filter by manual pressure on the syringe. A colorimetric detection allows for naked eye results interpretation. The assay provides sensitivity in the picomolar range in just 5 min, even using low volumes of sample in complex matrix. Probe deposition by spotting allows for flexible combinations of different capturing agents and multiple diagnoses; furthermore, the very simple and inexpensive set-up makes the syringe-based immunoassay on paper microarray a suitable diagnostic system for resource-limited settings.

Turn-on chemiluminescent sensing platform for label-free protease detection using streptavidin-modified magnetic beads.

We report a label-free streptavidin-modified magnetic beads (SA-MBs)-based sensing platform for turn-on chemiluminescent (CL) detection of protease using trypsin as model analyte. In the assay, a biotinylated peptide containing an arginine and a terminal cysteine was used as the substrate of trypsin. Upon adding the peptide into a basic luminol-NaIO4 solution, the terminal cysteine induced a strong CL signal. Surprisingly a much lower CL was emitted when the peptide was immobilized on the surface of SA-MBs. Based on this phenomenon, we designed a turn-on CL sensing system for protease using trypsin as model and its inhibitors screening. In the absence of trypsin, the peptide was coupled to the SA-MBs surface, resulting in a low CL background. Upon the addition of trypsin, the peptide can be catalytically hydrolyzed at the C-terminus of arginine, resulting in the formation of free cysteine-containing residues and subsequent CL recovery with the addition of luminol and NaIO4. The simple method does not require washing or separating procedures. Trypsin at a concentration as low as 10 pM can be assayed using this new CL sensing system. Additionally, the proposed method can be employed for screening the inhibitors of trypsin. This new sensing strategy could be easily extended to assay other proteases by simply changing the peptide substrate.

A simplified method to attach antibodies on liposomes by biotin-streptavidin affinity for rapid and economical screening of targeted liposomes.

The biotin-Streptavidin (STREP) technique for attachment of monoclonal antibodies (mAbs) (or other ligand types) on liposome surface offers high attachment yield, however it is time consuming and expensive due to the number of steps used and the consumption of large quantities of STREP. Herein, a simplified, fast and economic technique, by incubating pre-mixed biotin-mAb/STREP with biotin-liposomes, at a 3:1:1 biotin-mAb/STREP/biotin-LIP ratio (mol/mol/mol) was evaluated. The physichochemical properties, final mAb attachment yield and targeting potential of liposomes decorated with an anti-transferrin receptor mAb (TfR-mAb), prepared by the simple method (SM) and the conventional method (CM), were compared. The vesicle uptake by hCMEC/D3 cells (known to overexpress TfR) were considered as a measure of liposome targeting capability. Results show that both targeted liposome types (SM and CM) have small size (mean diameters around 150 nm), low poly-dispersity (approx. 0.20) and similar mAb attachment yield (between 64-88%). However, the uptake of the SM-liposomes is slightly lower compared to CM-LIP (24-30% decrease), suggesting that the modulated conformation of mAbs on the liposome surface (triplets attached to one single STREP molecule) results in decreased targeting capability. Nevertheless, the simpler and faster one-step preparation procedure which has very high lipid recovery (> 95%) compared to the CM (50-60%) and 15-30 times lower consumption of STREP, may be a good alternative for initial screening of various mAbs as ligands for targeted liposomal or other nanotechnologies, during pre-clinical development.

Fast magnetic isolation of simple sequence repeat markers in microfluidic channels.

Simple sequence repeat (SSR) markers are widely used for genome mapping, genetic diversity characterization and medical diagnosis. The fast isolation by AFLP of sequence containing repeats (FIASCO) is a powerful method for SSR marker isolation, but it is laborious, costly, and time consuming and requires multiple rounds of washing. Here, we report a superparamagnetic bead (SPMB)-based FIASCO method in a magnetic field controllable microfluidic chip (MFCM-Chip). This method dramatically reduces the assay time by 4.25-fold and reduces the quantity of magnetic beads and probes by 10-fold through the magnetic capture of (AG)n-containing fragments from Herba Leonuri, followed by washing and eluting on a microchip. The feasibility of this method was further evaluated by PCR and sequencing, and the results showed that the proportion of fragments containing SSRs was 89%, confirming that this platform is a fast and efficient method for SSR marker isolation. This cost-effective platform will make the powerful FIASCO technique more accessible for routine use with a wide variety of materials.

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

On-chip analysis of respiratory viruses from nasopharyngeal samples.

Point-of-care (PoC) testing followed by personalized efficient therapy of infectious diseases may result in a considerable reduction of associated health care costs. Lab-on-a-chip (LoC) systems represent a potentially high efficient class of PoC tools. Here, we present a LoC system for automated pathogen analysis of respiratory viruses from nasopharyngeal specimens. The device prepares total nucleic acids from extracted swab samples using magnetic silica beads. After reverse transcription the co-purified viral RNA is amplified in accordance with the QIAplex multiplex PCR technology. Hybridized to corresponding QIAGEN LiquiChip beads and labelled with streptavidin R-phycoerythrin, the amplified target sequences are finally detected using a QIAGEN LiquiChip200 workstation. All chemicals needed are either stored freeze-dried on the disposable chip or are provided in liquid form in a reagent cartridge for up to 24 runs. Magnetic stir bars for mixing as well as turning valves with metering structures are integrated into the injection-moulded disposable chip. The core of the controlling instrument is a rotating heating bar construction providing fixed temperatures for fast cycling. PCR times of about half an hour (for 30 cycles) could be achieved for 120 µl reactions, making this system the fastest currently available high-volume PCR chip. The functionality of the system was shown by comparing automatically processed nasopharyngeal samples to ones processed manually according to the QIAGEN "ResPlex™ II Panel v2.0" respiratory virus detection kit. A prototype of the present instrument revealed slightly weaker signal intensities with a similar sensitivity in comparison to the commercially available kit and automated nucleic acid preparation devices, even without protocol optimization.

Low-cost label-free biosensors using photonic crystals embedded between crossed polarizers.

There is a strong need for low-cost biosensors to enable rapid, on-site analysis of biological, biomedical, or chemical substances. We propose a platform for label-free optical biosensors based on applying the analyte onto a surface-functionalized photonic crystal slab and performing a transmission measurement with two crossed polarization filters. This dark-field approach allows for efficient background suppression as only the photonic crystal guided-mode resonances interacting with the functionalized surface experience significant polarization rotation. We present a compact biosensor demonstrator using a low-cost light emitting diode and a simple photodiode capable of detecting the binding kinetics of a 2.5 nM solution of the protein streptavidin on a biotin-functionalized photonic crystal surface.

A novel electrochemiluminescence strategy for ultrasensitive DNA assay using luminol functionalized gold nanoparticles multi-labeling and amplification of gold nanoparticles and biotin-streptavidin system.

Luminol functionalized gold nanoparticles were used as labels for electrochemiluminescence signal amplification and an ultrasensitive, highly selective, convenient, low cost DNA detection strategy was developed.

Laboratory-scale protein striping system for patterning biomolecules onto paper-based immunochromatographic test strips.

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.