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 of need, observe this operation under a microscope.</li>
	<li>Apply a small droplet of glue E6000 around the junction of the 90 &#181;m OD/100 &#181;m ID capillaries with the end of a Q-tip, and let the glue cure O/N at RT.</li>
	<li>Cut the inserted 20 &#181;m ID x 90 &#181;m OD capillary to a length of ~10-15 mm; this will be the electrospray ionization emitter.</li>
	<li>Pre-cut the 1/32&#34; OD PEEK, 1/16&#34; OD PEEK and 1/16&#34; OD PTFE tubing in pieces of 4-5 cm in length; these will be the sleeves used in capillary unions for providing a leak-free connection.</li>
	<li>Connect the opposite end of the 100 &#181;m ID x 360 &#181;m OD capillary to a PEEK union; use a 1/32&#34; PEEK sleeve for providing a leak-free connection.</li>
	<li>Insert/tighten a piece of ~5 cm long 1/16&#34; PTFE tubing into the opposite end of the union.</li>
	<li>Weigh ~4 mg of C18 (5 &#181;m) particles in a pre-cleaned/dried 2 ml glass vial; add 0.5 ml isopropanol, close the vial and disperse the slurry in the ultrasonicator bath.</li>
	<li>Withdraw with a 250 &#181;l syringe approximately 200 &#181;l slurry, insert the syringe needle in the 1/16&#34; PTFE tubing of the PEEK union, and dispense slowly the slurry into the 100 &#181;m ID x 360 &#181;m OD capillary; observe under the microscope as the capillary fills with particles until a length of 2-3 mm packing is achieved; this will be the microreactor (Figure 1). The 20 &#181;m ID capillary retains the particles in the microreactor through a Keystone effect.15</li>
	<li>Rinse the microreactor with ~50 &#181;l solution of H2O/CH3CN 50:50 v/v, and then with ~50 &#181;l solution of H2O/CH3CN 98:2 v/v.</li>
</ol>
2. Preparation of Sample Solutions
Note: Operations that involve handling of organic solvents and acids and solution preparation are to be performed in a fume hood. Wear goggles, gloves and protective clothing.
<ol>
	<li>Rinse with CH3OH and dry a few glass vials (4 ml) and polypropylene tubes (15 ml).</li>
	<li>Prepare 10 ml of a solution of H2O/CH3CN (98:2 v/v) in a 15 ml polypropylene tube by mixing 9.8 ml H2O with 200 &#181;l CH3CN; mix by sonication.</li>
	<li>Prepare 10 ml of a solution of H2O/CH3CN (50:50 v/v) in a 15 ml polypropylene tube by mixing 5 ml H2O with 5 ml CH3CN; mix by sonication.</li>
	<li>Prepare 4 ml of an acidified aqueous solution (TFA 0.01% v/v) by adding 4 &#181;l TFA(10%) to 4 ml H2O/CH3CN (98:2 v/v) in a 4 ml glass vial; mix by sonication.</li>
	<li>Prepare 4 ml of an acidified organic solution (TFA 0.01% v/v) by adding 4 &#181;l TFA(10%) to 4 ml H2O/CH3CN (50:50 v/v) in a 4 ml glass vial; mix by sonication.</li>
	<li>Weigh 16 mg NH4HCO3 with an analytical microbalance in a 4 ml glass vial; add 4 ml of the H2O/CH3CN (98:2 v/v) solution and disperse by sonication; this results in a 50 mM solution of NH4HCO3 (pH~7.8) in aqueous buffer.</li>
	<li>Weigh 5 mg of a standard protein (e.g., bovine serum albumin, hemoglobin &#945;/&#946;, cytochrome C, carbonic anhydrase, &#945;-2-HS-glycoprotein, etc.) with an analytical microbalance in a 4 ml glass vial; add 4 ml DI water and disperse by sonication; this typically results in a high &#181;M-level concentration solution.</li>
	<li>Prepare 1 ml protein solution (1-2 &#181;M) by diluting the high &#181;M-level concentration solution with an appropriate volume of NH4HCO3 (50 mM) aqueous buffer; mix by sonication.</li>
	<li>Prepare the trypsin solution (5 &#181;M) by dissolving 20 &#181;g sequencing-grade trypsin in 170 &#181;l NH4HCO3 (50 mM) aqueous buffer; disperse by sonication.</li>
</ol>
3. Experimental Setup&#160;
Note: The LTQ-MS system is fitted with a modified ESI source that includes a home-built XYZ-stage which enables interfacing of the mass spectrometer to various sample input approaches.
<ol>
	<li>Disconnect the capillary microreactor from the PEEK union and connect to the PEEK Tee.</li>
	<li>Insert a ~2 cm long Pt wire in the side-arm of the PEEK Tee; use a 1/32&#34; PEEK sleeve for providing a leak-free connection and for insulating the Pt wire.</li>
	<li>Connect a 50 &#181;m ID x 360 &#181;m OD (~0.5 m long) sample transfer capillary to the opposite end of the PEEK Tee; use a 1/32&#34; PEEK sleeve for providing a leak-free connection.</li>
	<li>Secure the PEEK Tee on the XYZ-stage and position the ESI emitter ~2 mm away from the mass spectrometer inlet capillary.</li>
	<li>Connect the Pt wire to the ESI power supply source of the mass spectrometer.</li>
	<li>Connect the opposite end of the sample transfer capillary to the stainless steel union; use a 1/16&#34; PEEK sleeve for providing a leak-free connection.</li>
	<li>Connect a 4-5 cm long 1/16&#34; PTFE tubing to the opposite end of the stainless steel union and insert the 250 &#181;l syringe needle in the PTFE tubing; turn on the pump and set the flow rate at the desired value (e.g., 2 &#181;l/min).</li>
	<li>Input tandem MS data acquisition parameters into the software package that controls the MS instrument and save as a method file to ready the setup for performing the analysis; for the LTQ-MS system, use the following data dependent analysis parameters: dynamic exclusion enabled for 180 sec with exclusion mass width &#177;1.5 m/z; one MS scan followed by one zoom and MS2 scans on the top 5 most intense peaks, zoom scan width &#177;5 m/z; collision induced dissociation (CID) parameters set at isolation width 3 m/z, normalized collision energy 35%, activation Q 0.25, and activation time 30 msec.</li>
</ol>
4. Microfluidic Setup&#160;
<ol>
	<li>Prepare a microfluidic device from glass or polymeric substrates;16-18 the layout of the device is provided in Figure 2A, consisting of a microreactor (1) (0.13 mm width x 2 mm length x 50 &#181;m deep) connected to an inlet (2), outlet (3) and a side port (4); the interconnecting channels for fluidic manipulations are ~110 &#181;m wide and ~50 &#181;m deep; a multichannel structure (5), consisting of ~1.5-2 &#181;m deep x 10 &#956;m wide x 100 &#956;m long microchannels placed 25 &#181;m apart, will act as a filter for retaining the particles in the microreactor.</li>
	<li>Enable fluid-delivery connections to the chip by gluing specialized connectors to the inlet ports, or by devising a polymeric stand that accommodates fittings for fluid delivery (Figure 2B).16</li>
	<li>Prepare a slurry of C18 particles (5 &#181;m) as described in step 1.8; deliver the slurry to the microreactor with a syringe by using 1/16&#34; PTFE tubing connected to the chip side port (4); seal the port after particle loading with epoxy glue and cure for 1 hr in an oven at 90 &#186;C.16</li>
	<li>Insert a capillary (20 &#181;m ID x 90 &#181;m OD x 10 mm length) in the outlet port, seal with E6000 glue, and let cure O/N; this will become the ESI emitter (6).</li>
	<li>Connect a 50 &#181;m ID x 360 &#181;m OD (~0.5 m long) sample transfer capillary to the chip inlet port (2); connect the opposite end of the capillary to a 250 &#181;l syringe and the syringe pump, as described in 3.6-3.7.</li>
	<li>Place a light-weight PEEK Tee connector on the sample transfer line just prior to the inlet port (2) and insert a Pt wire on the side-arm of the Tee, as described in 3.2; this will be the ESI electrode.</li>
	<li>Secure the microfluidic device on the XYZ stage with the ESI emitter positioned ~2 mm away from the mass spectrometer inlet capillary; prepare the MS for analysis as described in 3.8.</li>
</ol>
5. Sample Loading, Proteolytic Digestion and Elution for MS Analysis
Note: All solution/sample transfer steps are performed with the aid of a syringe pump and should allow for a few additional minutes to complete, to compensate for the dead-volumes associated with the transfer lines to and from the microreactor; the necessary time will depend on the flow rates involved.
<ol>
	<li>Rinse the microreactor and prepare it for analysis by infusing an aqueous solution of NH4HCO3 (50 mM) in H2O/CH3CN (98:2 v/v) at 2 &#181;l/min for 5 min; use a 250 &#181;l syringe.</li>
	<li>Load the protein sample (1 &#181;M) on the microreactor by infusing the sample solution at 2 &#181;l/min for 5 min; use a 250 &#181;l syringe.</li>
	<li>Infuse the trypsin solution (5 &#181;M) over the adsorbed protein on the microreactor at 2 &#181;l/min for 1-3 min.</li>
	<li>Quench the proteolytic digestion process by rinsing the microreactor with an acidified aqueous solution of TFA (0.01% v/v) in H2O/CH3CN (98:2 v/v) at 2 &#181;l/min for 5 min; use a 250 &#181;l syringe.</li>
	<li>Start eluting the protein digest from the microreactor by infusing an acidified organic solution of TFA (0.01% v/v) in H2O/CH3CN (50:50 v/v) at 300 nl/min.</li>
	<li>Turn on the MS data acquisition process and the ESI voltage (~2,000 V); acquire MS data using the data acquisition parameters provided in step 3.8; observe the elution of the tryptic peptides.</li>
</ol>
6. Data Processing
<ol>
	<li>Process the tandem MS data with a search engine compatible with the raw MS data format.
	<ol>
		<li>Process the LTQ-MS raw files using an organism specific minimally redundant protein database; upload the raw data in the search engine, set the parent and fragment ion mass tolerances to 2 and 1 Da, respectively, and use only fully tryptic fragments with up to two missed cleavages for the search; do not allow for posttranslational modifications; set the high and medium-confidence false discovery rates (FDR) to 1% and 3%, respectively, and do not allow for posttranslational modifications.</li>
	</ol>
	</li>
	<li>Filter the data to select only high-confidence peptide matches to the proteins in the database; evaluate the protein and peptide-level results.</li>
</ol>

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The authors declare no competing financial interests.

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), and can be accomplished within minutes from standard supplies, commonly found in a chromatography laboratory. While the reversed phase C18 particles can be rinsed with high organic-content solvent (CH3CN or CH3OH) for complete removal of adsorbed proteins or peptides, and the microreactor can be re-used, the easy and cost-effective fabrication process invites for the manufacturing of disposable units for single-use applications. The capillary microreactor can be mounted in a home-built ESI source, as shown in Figure 1B, or can be inserted in a standard commercial ion source. While the particle bed for sample capture and digestion does not need to be longer than ~1-2 mm, just enough to capture sufficient material for optimal detection by MS, the length of the capillary itself can be adjusted to fit the size of any particular ion source.
For laboratories that have capabilities to fabricate microfluidic devices, a simple design (Figure 2A) that can be secured into a stand for facilitating interfacing to MS detection (Figure 2B) is provided. The dimensions of the microreactor and transfer channels can be adjusted to the requirements of a particular application, and the device can be developed into a single or multiplexed format. For most commercial mass spectrometers, however, the ion source would need modifications to permit the placement of the device in the source. The photomask drawing for chip fabrication was executed with AutoCAD software and the photomask were prepared by HTA Photomask. The fabrication of the microchips was performed according to protocols described earlier:17,18 alignment of the substrate with the photomask, exposure to UV light (360 nm), removal of the irradiated photoresist and underlying chrome layer, wet chemical etching of channels with buffer oxide etch, removal of the remaining chrome layer, drilling of access holes, cleaning, hydrolysis of the microchip surface (NH4OH + H2O2), and thermal bonding of the substrate to the cover plate by gradual heating to 550 &#186;C. Both substrate and cover plate were etched, one for deep channels for sample handling (~20-50 &#181;m), and the other for shallow micro-channel filter elements (1.5-2 &#181;m deep).16
The results generated with the above-described microreactor are comparable with the results obtained from O/N,19 conventional digestion protocols that use substrate:enzyme ratios of (50-100):1, followed by a peptide infusion experiment with MS detection (300 nl/min, peptides dissolved in H2O/CH3CN (50:50 v/v) acidified with CH3COOH, 0.1 % v/v). Both microreactor and conventional protocols enabled the identification of all proteins by multiple unique peptides (Table 1). Typically, a somewhat larger number of unique peptides per protein were observable with the microreactor than with the conventional setup. This was a result of incomplete enzymatic cleavage at certain sites. A few additional minutes of proteolytic digestion reduced or eliminated this outcome. Also, some enzymatic peptides generated with the microreactor differed from the ones generated in solution, due to the fact that for the proteins adsorbed on the C18 particles different sites were exposed to the enzyme than in homogenous solutions.20 The Xcorr scores demonstrate, however, that the quality of the tandem mass spectra generated with the microreactor was of high-quality, and that a sufficient protein sequence coverage (11-75%) is achievable for unambiguous protein identifications.
The technique, as described, is limited to the analysis of rather simple protein mixtures that generate at most ~25-50 peptides that can be analyzed by simple infusion experiments and that do not necessitate liquid chromatographic separation prior to MS analysis. Critical to success is the use of freshly thawed and prepared trypsin solutions, to not reduce or lose the activity of the proteolytic enzyme at the operational pH of the microreactor (i.e., pH~8). In addition, a proper balance must be found between the optimal flow rates for electrospray ionization (150-300 nl/min) and the maximal flow rates that can be tolerated by the experimental setup and that enable fast peptide elution from the microreactor (2-3 &#181;l/min). Higher than optimal ESI flow rates result in sensitivity losses and poor detection limits. As a result, the length of the transfer capillaries should be maintained as short as possible to reduce the time necessary for the elution of peptides from the microreactor when operating at &#8804;300 nl/min. All components that were used for the experimental setup can be replaced by components from other manufacturers that perform similar functions. If the dimensional characteristics of these components differ (length, diameter, volume), the optimization of eluent flow rates, sample concentration, time for performing certain steps and of the ESI voltage may be necessary to obtain similar results.
A unique advantage enabled by the microfluidic setup is the ability to further incorporate on the chip a pumping system, e.g., an electroosmotic flow driven pump,21,22 for enabling the stand-alone operation of the platform and the integration of an additional separation step. The ability to perform on-chip separations of the peptides generated through proteolytic digestion will result in improved detection limits, and will facilitate the analysis of complex protein mixtures from cellular extracts. This will further enable the integration of the technique in workflows that make use of microfabricated platforms and high-speed MS detection for biomedical applications.23

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 number of missed cleavages, the peptide charge state (z), and the mass/charge (m/z) of the protonated peptides. At the protein level, the table provides the UniProt protein ID, the description (name) of the protein, the protein score and the protein coverage. All proteins were identifiable by multiple peptides, after a proteolytic digestion performed for 90 sec.
The time necessary for the elution of all peptides was dependent on the length of the transfer capillary that delivered the acidified organic solution at 300 nl/min, a flow rate compatible with nano-ESI optimal operation. In-capillary mixing of the aqueous and organic acidified solutions resulted in the early elution of a few peptides, but the great majority eluted over several minutes only in the organic solution. A mass spectrum displaying the co-elution of several peptides representative of the five proteins, generated with the capillary microreactor, is provided in Figure 3.
Table 1. List of tryptic peptides generated through the proteolytic digestion of five proteins using the capillary microreactor and O/N digestion protocols. Only unique sequences, with the highest Xcorr, are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53564/53564table1.xlsx" target="_blank">Please click here to download this file.</a>
<img alt="Figure 1" src="/files/ftp_upload/53564/53564fig1.jpg" /> 
Figure 1. Capillary microreactor. (A) Experimental setup for the fabrication of the microreactor. (B) Capillary microreactor positioned in the MS ion source. The microreactor is loaded manually with C18-bonded silica particles and placed on an XYZ-stage that facilitates proper positioning in the ESI-MS source. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53564/53564fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53564/53564fig2.jpg" /> 
Figure 2. Microfluidic microreactor. (A) Schematic diagram of the microfluidic reactor. (B) Microfluidic reactor secured in a PEEK stand. The microfluidic reactor and transfer lines are fabricated in a glass substrate which can be secured in a home-made polymeric PEEK stand for further positioning in the MS source with the XYZ stage. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53564/53564fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53564/53564fig3highres.jpg" width="700" /> 
Figure 3. Mass spectrum comprising tryptic peptides generated from the protein mixture subjected to proteolysis in the microreactor. The most intense tryptic peptide ions are labeled with the sequence of the amino acids and the corresponding protein identifier. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53564/53564fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.9K Views.  Virginia Tech. 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.

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

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 ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

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 ...

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 ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.