The authors wish to thank Zoiray Technologies Inc., a co-sponsor and commercialization partner, for its support.

1. Substrate Preparation
<ol>
<li>Coat layered silicon-SiO2 substrates with self-adsorbing copoly(DMA-NAS-MAPS):
 <ol>
 <li>Copolymer synthesis, chemical structure, and coating process are published in: G. Pirri, F. Damin, M. Chiari, E. Bontempi, L.E. Depero. Characterization of A Polymeric Adsorbed Coating for DNA Microarray Glass Slides. Anal. Chem. 2004, 76, 1352-58.</li>
 <li>Briefly, prepare polymer solution by adding 100 mg of polymer to 5 mL of deionized (DI) water and add 5 mL of 40% saturated ammonium sulfate ((NH4)2SO4) solution to reach a final concentration of 0.92 M.</li>
 <li>Submerge chips in solution for 30 min on a shaker and then rinse thoroughly with DI water. Dry thoroughly with Argon/N2 gas.</li>
 <li>Bake chips at 80 &deg;C for 15 min.</li> 
</ol>
</li>
<li>Store polymer-coated substrates/chips in a dry environment (vacuum desiccator) for up to 3 months until probe spotting procedure.</li>
</ol>
2. Preparation of Probe Array: Antibodies, antigens, ss/dsDNA, RNA, etc.
<ol>
	<li>Dilute probe(s) to appropriate concentration in desired buffer. This step can be vary considerably, but a typical experiment utilizes antigen or IgG at a concentration of 0.5 mg/mL (range of 0.1 -1 mg/mL) in phosphate buffered saline (PBS) at pH&asymp;7.4.</li>
	<li>Place solutions in a 96- or 384-well plate (or standard source plate for the spotter being used)</li>
	<li>Setup spotting parameters for desired printed array: determine the appropriate spotting parameters for the surface and solutions being used (dwell times, approach speeds, etc.). Determine the number of replicates of each condition per grid, the grid layout, the number of replicate grids, and the desired spotting location on the chip. Place substrates and source plate in the appropriate locations, check to make sure waste and supply bottles are ready, and begin printing run.</li>
	<li>After spotting is finished place substrates in a high humidity environment overnight (4-18 hours) to allow immobilization and deactivation process to proceed.</li>
	<li>Wash substrates: place them in the following solutions for three minutes for three separate washes: PBS with 0.1% Tween (PBST), PBS, and finally deionized (DI) water. Depending on the type of experiment (wet vs. dry), dry the chip thoroughly with Argon gas and begin incubation or place the chip in a flow chamber and assemble.</li>
	<li>Deactivate remaining NHS groups on copolymer surface by submerging the chips in a 50 mM solution of ethanolamine with the pH adjusted to 7.4 for 30 minutes while on a shaker plate. A primary amine-containing compound such as hydroxylamine or Tris can also be used, as long the prepared solution is safe to use with proteins. Thoroughly rinse the deactivation solution using PBS then DI water.</li>
	<li>Optional step (depending on the surface chemistry being employed): Block surface using a solution of BSA, casein, or rehydrated milk for 30 minutes. For the current polymeric surface chemistry that we use, we do not perform this step as the polymer backbone acts to prevent non-specific protein binding.</li>
</ol>
3. Data Acquisition and Incubation Procedure: Endpoint format
<ol>
<li>Make a solution of the target of interest in the desired buffer. Here, it will be a solution of the appropriate concentration of Anti-BSA in PBS (ex: make 1-10 mL of a 10 ng/mL solution of Anti-BSA in PBS). Also, make sure to have an equivalent amount of buffer (without target) for the negative control incubation.</li>
<li>Take a silicon mirror scan for normalizing all subsequently collected data.</li>
<li>Scan the prepared probe array with the IRIS system prior to the incubation step to determine the initial optical height (mass) at each spot. This will allow for proper analysis of changes in mass on the surface, i.e. the level of binding, after the incubation. Here, a few parameters will be adjusted for the current experiment, such as exposure time, field of view, prospering focusing, etc.</li>
<li>Submerge chip(s) in prepared solution for 1 hour on a shaker plate. The incubation time can vary considerably depending on the level of agitation, the concentration of target being detected, the transport characteristics of the flow chamber (if a real-time detection mode is being used), the affinities of the target-probe pair, etc.</li>
<li>After the incubation, repeat the wash procedure described in step 2.5)</li>
<li>Scan the same probe array using the IRIS system and obtain post-incubation images for pre-incubation comparison.</li>
<li>Repeat this process for different incubation steps if necessary, for example in a sandwich assay format where a secondary antibody is being used.</li>
</ol>
4. Data Analysis
<ol>
<li>Fit the collected images for each IRIS scan (sequence of intensity images), using the lab-developed software to produce an image of the probe array containing optical thickness information. A quick qualitative analysis of binding can be performed by subtracting (registered) post- and pre-incubation images. Binding will appear as a change in intensity at those spots where mass changes were observed. For quantitative analysis, determine mass densities of each spot compared to the background by averaging optical thickness information for each pixel within a spot and an annulus outside of the spot and making a direct comparison of these two areas.</li>
</ol>
5. Representative Results: 
Figure 1 shows an example schematic of the layered Si-SiO2 IRIS substrate spotted with a representative antibody array. Each antibody ensemble is spotted in replicate with specificity for a different epitope targeting different proteins. Two different whole viruses and a viral protein are represented as example targets. Negative control antibodies depend on the assay and can be non-specific and/or virus-specific.
Figure 2 shows the binding of human serum albumin (HSA)-specific antibodies to an array of spotted HSA and rabbit IgG (control) spots. As seen here, after fitting and determination of the optical thicknesses for the pre- and post-incubation images, a difference image for the binding array can be created to qualitatively assess binding. Quantitative analysis reveals that a 2.05 and 0.13 nanometer mean optical height change was observed for the HSA and rabbit IgG spots, respectively, for an anti-HSA incubation concentration of 500 ng/mL.
Figure 3 shows an IRIS measurement of Fur protein binding dependence on protein concentration and dsDNA oligomer length. The top graph shows absolute optical height measurements for initial immobilized oligomer probe heights and post-incubation Fur binding. Increasing concentrations of Fur (50, 100, 200 nM) resulted in increased binding to DNA probes. The length of the oligomers also impacts Fur binding with longer sequences resulting in more binding. Here, error bars represent one standard deviation from the mean. The bottom plot shows calculated Fur protein dimer binding per dsDNA strand. Dimer binding was significantly increased at a Fur protein concentration of 200 nM suggesting a high-level of non-specific binding.
<img src="/files/ftp_upload/2694/2694fig1.jpg" alt="Figure 1"/> 
Figure 1. Schematic of an example probe array normally used in an experiment to detect specific viruses and viral components in a multiplexed fashion with the IRIS system.
<img src="/files/ftp_upload/2694/2694fig2.jpg" alt="Figure 2"/> 
Figure 2. Post-incubation difference image for binding of human serum albumin-specific antibodies to spotted HSA collected with this label-free system at a concentration of 500 ng/mL. The biomaterial mass density for each spot is determined by averaging optical thickness information within a spot and then comparing this with the average of an annulus around the spot representing the background.
<img src="/files/ftp_upload/2694/2694fig3.jpg" alt="Figure 3"/> 
Figure 3. Plot of binding data for Fur protein interactions with spotted double-stranded oligomers with a known consensus sequence based on oligomer length, location of the consensus sequence within the oligomer, and Fur protein concentration. Information about the absolute amount of Fur protein bound to each spotted sequence (top) can be used to estimate the number of Fur dimers bound to each type of oligomer (bottom).

<ol>
	<li>&#214;zkumur, E., Needham, J. W., Bergstein, D. A., Gonzalez, R., Cabodi, M., Gershoni, J. M., Goldberg, B. B., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+and+dynamic+detection+of+biomolecular+interactions+for+high-throughput+microarray+applications.">Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications.</a> Proceedings of the National Academy of Sciences. 105, 7988-7992 (2008).</li><li>&#214;zkumur, E., Ahn, S., Yalcin, A., Lopez, C. A., Cevik, E., Irani, R. J., DeLisi, C., Chiari, M., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+microarray+imaging+for+direct+detection+of+DNA+hybridization+and+single-nucleotide+mismatches.">Label-free microarray imaging for direct detection of DNA hybridization and single-nucleotide mismatches.</a> Biosensors and Bioelectronics. 25, 1789-1795 (2010).</li><li>&#214;zkumur, E., Lopez, C. A., Yalcin, A., Bergstein, D. A., Connor, J. H., Chiari, M., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+reflectance+imaging+for+a+multiplexed,+high-throughput,+label-free,+and+dynamic+biosensing+platform.">Spectral reflectance imaging for a multiplexed, high-throughput, label-free, and dynamic biosensing platform.</a> IEEE Journal of Selected Topics in Quantum Electronics. 16, 635-646 (2010).</li><li>Lopez, C. A., Daaboul, G. G., Vedula, R. S., &#214;zkumur, E., Bergstein, D. A., Geisbert, T. W., Fawcett, H. E., Goldberg, B. B., Connor, J. H., &#220;nl&#246;, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+multiplexed+virus+detection+using+spectral+reflectance+imaging.">Label-free multiplexed virus detection using spectral reflectance imaging.</a> Biosensors and Bioelectronics. , (2011).</li></ol>

No conflicts of interest declared.

The IRIS platform is a simple and rapid system to use for collecting high-throughput binding data in a microarray format. By covalently immobilizing different functional protein or DNA probes on a surface, target antigens/biomarkers/etc. can be captured from solution, as in an immunoassay. Measurement of these interactions across probe conditions in an endpoint or real-time format with the IRIS system allows for highly sensitive and quantitative information to be collected for each interaction. The representative experiment detailed here is just one example of the types of interactions that can be studied with this technique. Detection of transcription factors, viral antigens, and whole viruses has been demonstrated previously and represents just a few examples of the types of analyses that the IRIS can easily handle.

<ol>
 <li>Silicon wafers with 500 nm of thermally-grown silicon dioxide, Silicon Valley Microelectronics, custom order
 <li> Ammonium sulfate powder, Sigma-Aldrich cat. # A5132
 <li> Phosphate buffered saline (PBS) 10X ready concentrate, Fisher Scientific cat. # BP665-1
 <li> Antibody to human serum albumin (Anti-HSA), Sigma-Aldrich cat. # A0433
 <li> Human serum albumin (HSA), Sigma-Aldrich cat. # A9511
 <li> Argon or nitrogen gas (ultra-high purity), Airgas cat. # AR UHP300 or # NI UHP300
 <li> Petri dishes, 60 mm x 15 mm, Fisher Scientific cat. # 0875713A
 <li> Scienceware&reg; vacuum desiccator, Sigma-Aldrich cat. # Z119016
 <li> Microcentrifuge tubes, Fisher Scientific cat. # 05-408-120
 <li> 96-well plates, low cell binding, Nalgene Nunc International cat. # 145399
 <li> BioOdyssey Calligrapher MiniArrayer, Bio-Rad
 <li> Tween-20, Sigma-Aldrich # P1379
 <li> Ethanolamine, Sigma-Aldrich # 398136
 <li> Hydroxylamine, Thermo Scientific cat. # 26103
 <li> Multi-purpose rotator, Thermo Scientific (Barnstead International), model # 2314, cat. # 2314Q
 <li> Retiga 2000R camera, QImaging cat. # 01-RET-2000R-F-M-12
 <li> AcuLED illumination source, PerkinElmer, custom order
 <li> Optical components (lenses, objectives, apertures, rails, posts, etc.), Thorlabs
</ol>

biomolecular detection employing the interferometric reflectance imaging sensor (iris)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Watch this Scientific Journal Video about Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS at JoVE.com

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

biomolecular-detection-employing-interferometric-reflectance-imaging

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JoVE Journal

Bioengineering

14.6K 조회수.  Boston University. 그런가에 대한 정량, 높은 처리량, 실시간 및 레이블이없는 biomolecular 감지 (DNA, 단백질 등) 2 표면은 LED 조명에 의존하고 간단한 interferometric 기술, 최소한의 광학 부품, 그리고 카메라를 사용하여 얻을 수 있습니다. Interferometric 반사율 이미징 센서 (IRIS)는 형식을 microarray로 저렴, 사용하기 간단하고, 의무가 있습니다.

비디오: Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.

In this study, the main purpose was to monitor the cellular uptake and eventual exocytosis of carbon nanotubes (CNTs). From this perspective, we proposed here a unique method using multispectral imaging flow cytometry allowing a quantification and localization of CNTs in a statistically relevant number of cells.

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Design and Fabrication of an Elastomeric Unit for Soft Modular Robots in Minimally Invasive Surgery

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in unstructured environments. At the same time, new procedures and techniques have been developed to reduce the invasiveness of surgical operations. Minimally Invasive Surgery (MIS) has been successfully employed for abdominal interventions, however standard MIS procedures are mainly based on rigid or semi-rigid tools that limit the dexterity of the clinician. This paper presents a soft and high dexterous manipulator for MIS. The manipulator was inspired by the biological capabilities of the octopus arm, and is designed with a modular approach. Each module presents the same functional characteristics, thus achieving high dexterity and versatility when more modules are integrated. The paper details the design, fabrication process and the materials necessary for the development of a single unit, which is fabricated by casting silicone inside specific molds. The result consists in an elastomeric cylinder including three flexible pneumatic actuators that enable elongation and omni-directional bending of the unit. An external braided sheath improves the motion of the module. In the center of each module a granular jamming-based mechanism varies the stiffness of the structure during the tasks. Tests demonstrate that the module is able to bend up to 120&#176; and to elongate up to 66% of the initial length. The module generates a maximum force of 47 N, and its stiffness can increase up to 36%.

This paper describes the design and fabrication of a soft unit for surgical manipulators. The base module includes three flexible fluidic actuators to achieve omnidirectional bending and elongation, and a granular jamming-based mechanism to enable stiffness control. A complete mechanical characterization is also reported.

In recent years, soft robotics technologies have aroused increasing interest in the medical field due to their intrinsically safe interaction in ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental monitoring, and food safety control. In this context, we used &#946;-lactamases as efficient reporter enzymes in several protein-protein interaction studies. Furthermore, their ability to accept insertions of peptides or structured proteins/domains strongly encourages the use of these enzymes to generate chimeric proteins. In a recent study, we inserted a single-domain antibody fragment into the Bacillus licheniformis BlaP &#946;-lactamase. These small domains, also called nanobodies, are defined as the antigen-binding domains of single chain antibodies from camelids. Like common double chain antibodies, they show high affinities and specificities for their targets. The resulting chimeric protein exhibited a high affinity against its target while retaining the &#946;-lactamase activity. This suggests that the nanobody and &#946;-lactamase moieties remain functional. In the present work, we report a detailed protocol that combines our hybrid &#946;-lactamase system to the biosensor technology. The specific binding of the nanobody to its target can be detected thanks to a conductimetric measurement of the protons released by the catalytic activity of the enzyme.

The Use of a &amp;#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

In this work, we report a new method to study protein-protein interactions using a conductimetric biosensor based on the hybrid &#946;-lactamase technology. This method relies on release of protons upon hydrolysis of &#946;-lactams.

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. pastoris. Small scale bioreactor cultivations (0.5-5 L working volume) are commonly used to evaluate a strain and its protein production characteristics since microscale cultivation in deep well plates can be hardly controlled or relies on expensive equipment. Furthermore, traditional protocols for the cultivation and induction of P. pastoris were established for constitutive expression or methanol induction and so far, no reliable protocols were described to screen P. pastoris expression strains with derepressible promoters in (controlled and monitored) parallel cultivations. To simplify such initial cultivations to characterize and compare new protein production strains, we established a simple shake flask cultivation system for methanol free expression that simulates bioreactor conditions including a constant slow glycerol feed and online monitoring, thereby coming closer to the real conditions in bioreactors compared to mostly applied small scale batch cultivations. To drive recombinant protein expression in P. pastoris, the carbon source repressed promoters PDC and PDF were applied. Polymer discs with embedded carbon source, releasing a constant amount of glycerol, assured a feed rate delivering the necessary energy for maintaining the promoters active while keeping the biomass generation low.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab ...

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10&#8211;60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.

Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution.

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...