Name: fast enzymatic processing of proteins for ms detection with a flow-through microreactor
Uploaded: 2016-04-06T13:00:00
Description: Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

This work was supported by NSF/DBI-1255991 grant to IML.

1. Preparation of the Capillary Microreactor
<ol>
	<li>Cut the 100 &#181;m internal diameter (ID) x 360 &#181;m outer diameter (OD) capillary to a length of 7-8 cm, and the 20 &#181;m ID x 90 &#181;m OD capillary to 3-5 cm with the glass capillary cleaver; verify under the microscope that both capillary ends have a clean, straight cut, without any protruding burrs.</li>
	<li>Insert the 20 &#181;m ID x 90 &#181;m OD capillary into one end of the 100 &#181;m ID x 360 &#181;m OD capillary, for a length of ~6 mm; in case o...

<ol>
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The microreactor described in this work provides an easy-to-implement experimental setup for performing enzymatic digestion of proteins to enable MS analysis and identification in less than 30 min. The distinct advantages of this system, in comparison to conventional approaches, include simplicity, speed, low reagent consumption and low costs. In particular, there is no need for expensive immobilized trypsin beads and cartridges. The preparation of the capillary microreactor is straightforward (Figure 1A...

The identification and characterization of purified proteins is frequently achieved by using MS techniques. The protein is digested with an enzyme and its peptides are further analyzed by MS by using a simple infusion experimental setup. Proteolytic digestion is necessary for generating small peptide fragments that fall in the useful mass range of most MS analyzers, and that can be easily fragmented through low energy collision induced dissociation to generate amino acid sequence information. For isolated proteins or simple protein mixtures, there is no further need for chromatographic separation of peptides prior to MS detection. A mixture of 25-50 peptides can be easily analyzed by infusing the sample with a syringe pump directly in the MS ion source.
The mass spectrometer can perform the analysis and confirm the sequence of a protein within a short time-frame. With modern data acquisition methods, this process can be accomplished within a few minutes or even seconds. The limiting factor in completing the entire process on a short time-scale is the proteolytic digestion step. Typically, this is performed over a few hours (or O/N), in solution, at 37 &#186;C, using substrate:enzyme ratios of (50-100):1. To reduce the enzymatic digestion time to minutes or seconds, immobilized enzyme microreactors, in the form of microfluidic reactors or commercially available cartridges, have been described.1-6 Typically, the enzyme is immobilized by covalent, non-covalent/physical adsorption, complex formation or encapsulation,3,6 the enhanced efficiency of the enzymatic process being enabled by the large surface-to-volume and enzyme-to-substrate ratios. Additional advantages of immobilized reactors include reduced autolysis and interference from the enzyme in MS analysis, improved enzyme stability and reusability. A variety of approaches, using glass or polymeric microfabricated devices have been described, using enzymes immobilized on magnetic beads by antibody-antigen interactions,7,8 entrapped in gold nanoparticle networks,9 encapsulated in titania-alumina sol-gels10 and nanozeolites,11 or captured through Ni-NTA or His-Tag complex formation.6 Alternatively, open-tubular capillaries with immobilized enzymes have been developed, as well.12 Moreover, enhanced proteolytic cleavage has been demonstrated by using controlled microwave irradiation13 or pressure-assisted or pressure cycling technology (PCT) for reducing the reaction times to 30-120 min.14
Despite the multiple advantages of immobilized enzyme reactors, the costs of commercial cartridges is high, the availability of microfluidic devices for routine use is limited, and the use of microwave or PCT technologies results in need for additional instrumentation. The goal of this work was to develop a method that circumvents these disadvantages, and that can be easily implemented in every laboratory to empower researchers with a simple and effective approach for performing enzymatic cleavage of proteins in preparation for MS analysis within minutes. The approach relies on the use of hydrophobic, C18-particles which are pre-loaded in a capillary or microfluidic device, and the adsorption of the protein(s) of interest on these particles followed by enzymatic digestion during the infusion of the enzyme over the packed bed and captured protein(s). In this approach, the substrate is immobilized through non-covalent interactions, and the enzyme is infused over the immobilized protein. The proteolytic digestion efficiency is increased by the large particle surface areas that expose the protein for enzymatic processing, reduced distances and diffusion times to and from the surface of particles, improved mass transfer, no covalent attachment that may affect the activity of the enzyme, ability to quickly evaluate combinations of different enzymes, disposability, and multiplexing if the process is executed in a microfluidic format. This approach is demonstrated with the use of a mixture of standard proteins and trypsin-the most commonly used enzyme for proteolytic digestion prior to ESI-MS detection. The mass spectrometer used for detection in this study was a linear trap quadrupole (LTQ) instrument.

<table>
	<tbody>
		<tr>
			<td>Ion trap ESI-MS</td>
			<td>Thermo Electron</td>
			<td>LTQ</td>
			<td>The LTQ mass spectrometer is used for acquiring tandem MS data</td>
		</tr>
		<tr>
			<td>XYZ stage</td>
			<td>Newport</td>
			<td>Multiple parts</td>
			<td>The home-built XYZ stage is used to adapt the commercial LTQ nano-ESI source to receive input from various sample delivery systems</td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Edmund optics</td>
			<td>G81-278</td>
			<td>The microscope is used to observe the microreactor packing process</td>
		</tr>
		<tr>
			<td>Analytical balance/Metler</td>
			<td>VWR</td>
			<td>46600-204</td>
			<td>The balance is used to weigh the protein samples</td>
		</tr>
		<tr>
			<td>Ultrasonic bath/Branson</td>
			<td>VWR</td>
			<td>33995-540</td>
			<td>The sonic bath is used for mixing/homogenizing the samples and dispersing the C18 particle slurry</td>
		</tr>
		<tr>
			<td>Syringe pump 22</td>
			<td>Harvard Apparatus</td>
			<td>552222</td>
			<td>The micropump is used for loading, rinsing and eluting the sample and the enzyme on and from the packed capillary microreactor</td>
		</tr>
		<tr>
			<td>Milli-Q ultrapure water system&#160;</td>
			<td>EMD Millipore</td>
			<td>ZD5311595&#160;</td>
			<td>The MilliQ water system is used to prepare purified DI water</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (1000 &#181;L)</td>
			<td>VWR</td>
			<td>53513-410</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (100 &#181;L)</td>
			<td>VWR</td>
			<td>53513-406</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (10 &#181;L)</td>
			<td>VWR</td>
			<td>53513-402</td>
			<td>The pipettor is used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Fused silica capillary (100 &#181;m ID x 360 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP100375</td>
			<td>This capillary is used for the fabrication of the microreactor</td>
		</tr>
		<tr>
			<td>Fused silica capillary (20 &#181;m ID x 100 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP020090</td>
			<td>This capillary is used for the fabrication of the ESI emitter</td>
		</tr>
		<tr>
			<td>Fused silica capillary (50 &#181;m ID x 360 &#181;m OD)</td>
			<td>Polymicro Technologies</td>
			<td>TSP050375</td>
			<td>This capillary is used to transfer the samples and the eluent from the syringe pump to the capillary microreactor</td>
		</tr>
		<tr>
			<td>Glass capillary cleaver</td>
			<td>Supelco</td>
			<td>23740-U</td>
			<td>This is a tool for cutting fused silica capillaries at the desired length</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>Eclectic Products</td>
			<td>E6000 Craft</td>
			<td>This glue is used for securing the ESI emitter into the capillary microreactor or the microfluidic chip</td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Epo-Tek</td>
			<td>353NDT</td>
			<td>This glue is used to seal the microfluidic inlet hole through which the C18 particles are loaded</td>
		</tr>
		<tr>
			<td>Reversed phase C18 particles (5 &#181;m)</td>
			<td>Agilent Technologies</td>
			<td>Zorbax 300SB-C18</td>
			<td>These are C18 particles on which the proteins are adsorbed; the particles were extracted from a 4 mm x 20 cm C18 LC column from Agilent</td>
		</tr>
		<tr>
			<td>Syringe/glass (250 &#181;L)</td>
			<td>Hamilton</td>
			<td>81130-1725RN</td>
			<td>The glass syringes are used to load the C18 particle slurry in the capillary microreactor and to deliver the sample and eluents to the microreactor</td>
		</tr>
		<tr>
			<td>Internal reducing PEEK Union (1/16&#8221; to 1/32&#8221;)</td>
			<td>Valco</td>
			<td>ZRU1.5FPK</td>
			<td>This union is used to connect the 250 &#181;L syringe to the microreactor for loading the 5 &#181;m particle slurry</td>
		</tr>
		<tr>
			<td>Stainless steel union (1/16&#8221;)</td>
			<td>Valco</td>
			<td>ZU1XC</td>
			<td>The stainless steel union is used to connect the glass syringe needle to the infusion capillary</td>
		</tr>
		<tr>
			<td>Microvolume PEEK Tee connector (1/32&#8221;)</td>
			<td>Valco</td>
			<td>MT.5XCPK</td>
			<td>The Peek tee is used to connect the sample transfer capillary to the capillary microreactor; on its side arm, it enables the insertion of the Pt wire</td>
		</tr>
		<tr>
			<td>Tee connector (light weight)</td>
			<td>Valco</td>
			<td>C-NTXFPK</td>
			<td>This Tee connector is used to apply ESI voltage to the microfluidic chip through the sample transfer line</td>
		</tr>
		<tr>
			<td>Pt wire (0.404 mm)</td>
			<td>VWR</td>
			<td>66260-126</td>
			<td>The Pt wire provides electrical connection for ESI generation and is connected to the mass spectrometer ESI power supply</td>
		</tr>
		<tr>
			<td>PTFE tubing (1/16&#8221; OD)</td>
			<td>Valco</td>
			<td>TTF115-10FT</td>
			<td>The Teflon tubing is used to enable an air-tight connection between the syringe needle and the stainless steel union&#160;</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8220; ID x 1/16&#8221; OD)</td>
			<td>Upchurch Scientific</td>
			<td>1565</td>
			<td>The Peek tubing is used as a sleeve to enable an air-tight connection between the stainless steel union and the 50 &#181;m ID transfer capillary</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8221; ID x 1/32&#8221; OD)</td>
			<td>Valco</td>
			<td>TPK.515-25</td>
			<td>The Peek tubing is used as a sleeve to enable a leak-free connection between the fused silica capillaries and the Peek Tee</td>
		</tr>
		<tr>
			<td>Clean-cut polymer tubing cutter</td>
			<td>Valco</td>
			<td>JR-797</td>
			<td>This cutter is used to pre-cut the 1/16&#8221; and 1/32&#8217; Peek polymer tubing that is used as sleeve for leak-free connections in pieces of ~4-5 cm in length</td>
		</tr>
		<tr>
			<td>Amber vial (2 mL)</td>
			<td>Agilent&#160;</td>
			<td>HP-5183-2069</td>
			<td>The vials are used to prepare sample solutions and the C18 particle slurry&#160;</td>
		</tr>
		<tr>
			<td>Amber vial (4 mL)</td>
			<td>VWR</td>
			<td>66011-948</td>
			<td>The vials are used to prepare sample solutions</td>
		</tr>
		<tr>
			<td>Polypropylene tube (15 mL)</td>
			<td>Fisher</td>
			<td>12-565-286D</td>
			<td>The vials are used to prepare buffer solutions</td>
		</tr>
		<tr>
			<td>Cylinder (100 mL)</td>
			<td>VWR</td>
			<td>24710-463</td>
			<td>The cylinder is used to measure volumes of solvent</td>
		</tr>
		<tr>
			<td>Cylinder (10 mL)</td>
			<td>VWR</td>
			<td>24710-441</td>
			<td>The cylinder is used to measure volumes of solvent</td>
		</tr>
		<tr>
			<td>Pipette tips (1000 &#181;L)</td>
			<td>VWR</td>
			<td>83007-386</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipette tips (100 &#181;L)</td>
			<td>VWR</td>
			<td>53503-781</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Pipette tips (10 &#181;L)</td>
			<td>VWR</td>
			<td>53511-681</td>
			<td>The pipette tips are used to measure small volumes of sample solutions</td>
		</tr>
		<tr>
			<td>Glass substrates</td>
			<td>Nanofilm</td>
			<td>B270 white crown, 3&#8221; x 3&#8221;</td>
			<td>These are glass substrates for microchip fabrication</td>
		</tr>
		<tr>
			<td>Male nut fitting (1/16&#8221;)</td>
			<td>Upchurch</td>
			<td>P203X</td>
			<td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td>
		</tr>
		<tr>
			<td>Nanoport assembly</td>
			<td>Upchurch</td>
			<td>N-122H</td>
			<td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protein standards</td>
			<td>Sigma</td>
			<td>Multiple #</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, HPLC grade</td>
			<td>Fisher</td>
			<td>A955</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC grade</td>
			<td>Fisher</td>
			<td>A452</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol, HPLC grade</td>
			<td>Sigma</td>
			<td>650447</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Aldrich</td>
			<td>A6141</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, sequencing grade</td>
			<td>Promega</td>
			<td>V5111</td>
			<td></td>
		</tr>
	</tbody>
</table>

fast enzymatic processing of proteins for ms detection with a flow-through microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A representative result of a proteolytic digestion process performed simultaneously on a mixture of proteins, with the above-described microreactors (Figures 1 or 2), is provided in Table 1. The table comprises the unique peptide sequences that identify a particular protein, the cross correlation score (Xcorr) (i.e., a score that characterizes the quality of the experimental-to-theoretical match for the corresponding tandem mass spectrum), the nu...

Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

The overall goal of this protocol is to describe the fabrication of a flow-through tryptic mircoreactor that performs fast enzymatic digestion of proteins for enabling protein identification with an electrospray ionization mass spectrometer. The method can help illustrate the amino acid sequence of isolated and purified proteins to support downstream characterization of structure and function. The main advantage of this protocol is that it is fast, cost-effective, and prone for integration into workflows that make use of microfabricated platforms.To begin, use a glass capillary cleaver to cut the larger glass capillary tube to a length of seven to eight centimeters, and the smaller one to a length of three to five centimeters. Use a microscope to verify that both capillary ends have a clean, straight cut, without any protruding burrs. Insert the smaller capillary about six millimeters into one end of the larger one.Then, use a Q-tip to apply a small droplet of glue around the junction of the capillaries, and let the glue cure overnight at room temperature. Next, cut the inserted capillary to a length of about 10 to 15 millimeters. This end will act as the electrospray ionization emitter.Insert the opposite end of the larger diameter capillary into a 1/32-inch PEEK tube that has been pre-cut between four and five centimeters in length and connected to a PEEK union. Then, insert and tighten the five centimeter-long piece of 1/16 PTFE tubing into the opposite end of the union. In a clean, dry two milliliter glass file, weight out four milligrams of C18 particles and then add 0.5 milliliters of isopropanol.Close the vial. Place it in ultrasonicator bath and sonicate to disperse the particles evenly in the solution. Next, use a 250 microliter syringe to draw up 200 microliters of the C18 slurry.Insert the syringe into the 1/16-inch PTFE tubing and dispense the slurry slowly into the large capillary. Observe under the microscope as the capillary fills with particles, until two to three millimeters of particle packing has been achieved. The smaller capillary tube retains these particles in the microreactor through a keystone effect.Finally, rinse the microreactor with 50 microliters of a 50-to-50 solution of water and acetonitrile, followed by 50 microliters of a solution containing 49 microliters of water, mixed with one microliter of acetonitrile. Disconnect the capillary microreactor from the PEEK union and connect it to a PEEK-T. Then, insert a two centimeter-long platinum wire, insulated with a 1/32-inch PEEK sleeve to provide a leak-free connection into the side arm of the PEEK-T.Connect a half a meter-long sample transfer capillary to the opposite end of the PEEK-T. Use a 1/32-inch PEEK sleeve to provide a leak-free connection. Secure the PEEK-T on the XYZ stage.And position the electrospray ionization emitter about two millimeters away from the mass spectrometer inlet capillary. Then, connect the platinum wire to the electrospray ionization power supply source of the mass spectrometer. Also, connect the opposite end of the sample transfer capillary to the stainless steel union, using a 1/16-inch PEEK sleeve to provide a leak-free connection.Next, connect a four to five centimeter-long piece of 1/16-inch-long PTFE tubing to the opposite end of the stainless steel union. Input the tandem MS data acquisition parameters as described in the accompanying text protocol into the software package that controls the MS instrument. Save them as a method file to ready the setup for performing the analysis.The LTQ MS system is fitted with a modified ESI source that includes a home-built XYZ stage, which enables the interfacing of mass spectrometer to the various sample input approaches. Load a 250 microliter syringe with an aqueous solution of 50 millimolar bicarbonate dissolved in 2%of acetonitrile. Then, connect the syringe to the microreactor, and place it under the syringe pump.Rinse the microreactor and two microliters per minute for five minutes, to prepare it for analysis. Then, load the protein sample of interest into a 250 microliter syringe and infuse the sample solution at two microliters per minute for five minutes. Next, place a 250 microliter syringe loaded with a five micromolar trypsin solution under the syringe pump, and infuse over the absorbed protein on the microreactor at two microliters per minute for one to three minutes.It's critical that one freshly thaws and prepares the trypsin solution before each experiment. This will ensure that the protolytic enzyme retains its activity and its operational pH of the microreactor, which is a pH of about 8. Load another 250 microliter syringe with an aqueous solution consisting of 0%1%of trifluoroacetic acid added to 2%acetonitrile.Use this solution to quench the proteolytic digestion process by rinsing the microreactor at two microliters per minute for five minutes. Then, switch to a syringe filled with 01%trifluoroacetic acid added to 50%acetonitrile, and turn on the MS Data Acquisition Process in the Electrospray Ionization voltage to about 2000 volts. Use the acidified solution to start eluting the protein digest from the microreactor at 300 nanoliters per minute for 20 to 30 minutes.Acquire mass spec data using the data acquisition parameters provided in the accompanying text protocol. Process the LTQ MS raw files using a minimally redundant protein database by first uploading the raw data in the search engine. Then, set the parent and fragment ion mass tolerances to two and one daltons respectively, and use only fully tryptic fragments with up to two missed cleavages for the search.In addition, set the high and medium confidence false discovery rates to 1%and 3%respectively, and do not allow for post-translational modifications, unless specifically looking for particular modifications. Finally, filter the data to select only the high confidence peptide matches. Shown here is a mass spectrum comprised of tryptic peptides that were generated from a protein mixture that was subjected to proteolysis in the microreactor.These labels represent the most intense tryptic peptide ions and provide their sequence and corresponding protein identifier. This microreactor provides an easy-to-implement experiment set up for performing enzymatic digestion of proteins and enables a mass analysis and identification in less than 30 minutes. While attempting this procedure, it is important to remember that preserving the cleanliness of the vials and instrumentation that come into contact with solutions is critical to avoiding sample contamination.Following this procedure, other mass spectrometry data acquisition methods can be used to enable a better characterization of the generative peptides and answer additional questions related to structure of proteins. After its development, this technique paved the way for researchers in the field of proteonomics to develop faster protocols for the analysis of proteins and explore the development of novel microphalytic platforms that enable the high throughput exploration of biological samples. This technique is limited to the analysis of rather simple protein mixtures that generate 25 to 50 peptides.These peptides can be analyzed by simple infusion experiments and do not necessitate liquid chromatographics separation prior to MS analysis. Don't forget that working with organic solvents can be extremely hazardous, and precautions such as avoiding open flames should always be taken while performing this procedure.

fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Research

JoVE Journal

Bioengineering

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. ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

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 ...

Fast Pyrolysis of Biomass Residues in a Twin-screw Mixing Reactor

Fast pyrolysis is being increasingly applied in commercial plants worldwide. They run exclusively on woody biomass, which has favorable properties for conversion with fast pyrolysis. In order to increase the synergies of food production and the energetic and/or material use of biomass, it is desirable to utilize residues from agricultural production, e.g., straw. The presented method is suitable for converting such a material on an industrial scale. The main features are presented and an example of mass balances from the conversion of several biomass residues is given. After conversion, fractionated condensation is applied in order to retrieve two condensates &#8212; an organic-rich and an aqueous-rich one. This design prevents the production of fast pyrolysis bio-oil that exhibits phase separation. A two phase bio-oil is to be expected because of the typically high ash content of straw biomass, which promotes the production of water of reaction during conversion.
Both fractionated condensation and the use of biomass with high ash content demand a careful approach for establishing balances. Not all kind of balances are both meaningful and comparable to other results from the literature. Different balancing methods are presented, and the information that can be derived from them is discussed.

A procedure for thermochemical conversion of biomass residues is presented that aims at maximizing the yield of liquid products (fast pyrolysis). It is based on a technology proven on an industrial scale and especially suitable for treating a straw type of biomass.

Fast pyrolysis is being increasingly applied in commercial plants worldwide. They run exclusively on woody biomass, which has favorable properties for ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...