Development of a semi-automated MHC-associated peptide proteomics (MAPPs) method using streptavidin bead-based immunoaffinity capture and nano LC-MS/MS to support immunogenicity risk assessment in drug development.

Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an ex vivo method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semi-automated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment, in vitro/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads.

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.

All-in-one calcium nanoflowers for dual outputs biosensor: A simultaneous strategy for depression drug evaluation and non-invasive stress assessment.

Although enzyme-based signal amplification has been well developed for biosensors, their application in low-abundance biomarker under complicated conditions detection remains challenge. Cortisol is a steroid hormone and a quantitative evaluation of cortisol can objectively assess stress and depression. However, various factors can induce slight cortisol changes in body fluids, and this in turn sets a strict requirement for bedside testing of cortisol for evaluation of stress. Herein, all-in-one calcium nanoflowers (CaHPO4-AM-HRP-SA NFs) integrated with horseradish peroxidase (HRP), α-amylase (α-AM), and streptavidin (SA) have been synthesized to develop a simple but powerful biosensor for cortisol detection. High specific surface area and allosteric modulator provided by the hybrid nanoflowers as inherent advantages significantly boosted the catalytical ability and stability compared with the free enzymes. CaHPO4-AM-HRP-SA NFs also endowed the sensor with two output signals of one sample, leading the as-prepared sensor to realize self-calibration detection. Aside from using a traditional microplate reader to measure the signal, it could also be read out by a handheld blood glucose meter and a mobile phone. The sensor exhibited attractive simplicity and sensitivity with a low LOD of 98.5 pg mL-1. It accomplished the sensitive evaluation of cortisol in rat serum and assessed the antidepressant effects of different medications. The non-invasive and reliable cortisol detection is also achieved in human urine and saliva samples. Overall, we have demonstrated that the sensor can be deployed as a promising platform to evaluate drug efficiency and monitor stress in a simple and non-invasive manner.

Universal Surface Biotinylation: a simple, versatile and cost-effective sample multiplexing method for single-cell RNA-seq analysis.

Recent advances in single-cell analysis technology have made it possible to analyse tens of thousands of cells at a time. In addition, sample multiplexing techniques, which allow the analysis of several types of samples in a single run, are very useful for reducing experimental costs and improving experimental accuracy. However, a problem with this technique is that antigens and antibodies for universal labelling of various cell types may not be fully available. To overcome this issue, we developed a universal labelling technique, Universal Surface Biotinylation (USB), which does not depend on specific cell surface proteins. By introducing biotin into the amine group of any cell surface protein, we have obtained good labelling results in all the cell types we have tested. Combining with DNA-tagged streptavidin, it is possible to label each cell sample with specific DNA 'hashtag'. Compared with the conventional cell hashing method, the USB procedure seemed to have no discernible adverse effect on the acquisition of the transcriptome in each cell, according to the model experiments using differentiating mouse embryonic stem cells. This method can be theoretically used for any type of cells, including cells to which the conventional cell hashing method has not been applied successfully.

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.

Assessment of Streptavidin Bead Binding Capacity to Improve Quality of Streptavidin-based Enrichment Studies.

The streptavidin-based enrichment of biotin-tagged molecules is a common methodology that is routinely used across multiple disciplines in biomedical research. Numerous and varied formats of immobilized streptavidin and related proteins are available, but predicting which product is most apt for a given application is complicated by the fact that there are numerous technical considerations and no universal reporting standards for describing the binding capacity of the beads. Here, we define criteria that should be considered when performing a fit-for-purpose evaluation of streptavidin beads. We also describe a colorimetric competitive displacement assay, the streptAVIdin binDing capacITY (AVIDITY) assay, a fast, easy, and inexpensive absorbance-based method to measure the binding capacity of streptavidin beads, which can be used to compare different products and evaluate variation among many of the same product. We expect that the fit-for-purpose criteria and the AVIDITY assay will benefit users across disciplines to make informed decisions regarding the most apt streptavidin bead products for their own experiments.

Demonstration of a Label-Free and Low-Cost Optical Cavity-Based Biosensor Using Streptavidin and C-Reactive Protein.

An optical cavity-based biosensor (OCB) has been developed for point-of-care (POC) applications. This label-free biosensor employs low-cost components and simple fabrication processes to lower the overall cost while achieving high sensitivity using a differential detection method. To experimentally demonstrate its limit of detection (LOD), we conducted biosensing experiments with streptavidin and C-reactive protein (CRP). The optical cavity structure was optimized further for better sensitivity and easier fluid control. We utilized the polymer swelling property to fine-tune the optical cavity width, which significantly improved the success rate to produce measurable samples. Four different concentrations of streptavidin were tested in triplicate, and the LOD of the OCB was determined to be 1.35 nM. The OCB also successfully detected three different concentrations of human CRP using biotinylated CRP antibody. The LOD for CRP detection was 377 pM. All measurements were done using a small sample volume of 15 µL within 30 min. By reducing the sensing area, improving the functionalization and passivation processes, and increasing the sample volume, the LOD of the OCB are estimated to be reduced further to the femto-molar range. Overall, the demonstrated capability of the OCB in the present work shows great potential to be used as a promising POC biosensor.

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.

Magnetic Nanoparticle-Based Automatic Chemiluminescent Enzyme Immunoassay for Golgi Protein 73 and the Clinical Assessment.

Golgi protein 73 (GP73) is an independent diagnostic indicator of cirrhosis. However, it lacks automatic detection techniques to meet the large-scale clinical requirement in physical examination. In this paper, an automatic approach was established based on the ACL2800 automatic chemiluminescent analyzer, using magnetic nanoparticles (MNPs) and chemiluminescence. This method depended on sandwich strategy among biotin-labeled capture monoclonal antibody, target GP73 and acridinium ester (AE)-labeled reporter monoclonal antibody, conjugation of streptavidin-labeled MNPs to biotin-labeled capture monoclonal antibody, and chemiluminescent detection of AE-linked targets. Optimal conditions were investigated and clinical assessment was processed. Detection of GP73 demonstrated a high sensitivity of 1.19 ng/mL with a wide range from 1.34 ng/mL to 684.38 ng/mL. The quantitative detection was achieved with the repeatability of 2.69%, the coefficient of variation of 3.55% and the percent recovery of 93.46%-107.82%. Therefore, an automatic quantitative detection method was successfully developed, which was a potential screening method in physical examination.

Label-free real-time detection of biotinylated bovine serum albumin using a low-cost optical cavity-based biosensor.

We have developed a low-cost optical cavity-based biosensor with a differential detection method for point-of-care medical diagnostics. To experimentally demonstrate its label-free real-time biosensing capability, we performed the detection of biotinylated bovine serum albumin (BSA). Streptavidin is introduced into the optical cavity structure and immobilized on 3-aminopropyltriethoxysilane (APTES) coated surface. After rinsing out unbound streptavidin with DI water, biotinylated BSA without any labeling is introduced. A CMOS camera captures the transmitted light of two different wavelengths passing through the optical cavity sensing area in real-time. Then, the differential values are calculated to enhance the responsivity. We successfully demonstrated the label-free real-time detection of biotinylated BSA, and the measurement results matched well with the simulation results. The limit of detection of the optical cavity-based biosensor for the biotinylated BSA detection with the sensing area of 180 µm × 180 µm is estimated to be 2.82 pM, which could be reduced further for a smaller sensing area with the tradeoff of a longer sensing time.

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.

Simultaneous multi-species tracking in live cells with quantum dot conjugates.

Quantum dots are available in a range of spectrally separated emission colors and with a range of water-stabilizing surface coatings that offers great flexibility for enabling bio-specificity. In this study, we have taken advantage of this flexibility to demonstrate that it is possible to perform a simultaneous investigation of the lateral dynamics in the plasma membrane of i) the transmembrane epidermal growth factor receptor, ii) the glucosylphospatidylinositol-anchored protein CD59, and iii) ganglioside GM1-cholera toxin subunit B clusters in a single cell. We show that a large number of the trajectories are longer than 50 steps, which we by simulations show to be sufficient for robust single trajectory analysis. This analysis shows that the populations of the diffusion coefficients are heterogeneously distributed for all three species, but differ between the different species. We further show that the heterogeneity is decreased upon treating the cells with methyl-ß-cyclodextrin.