The authors would like to thank John Grazul (Cornell University), Andre Marziali (The University of British Columbia at Vancouver) and Vincent Tabard-Cossa (The University of Ottawa) for their advice. This work is funded in part by grants from the US National Science Foundation (DMR-0706517 and DMR-1006332) and the National Institutes of Health (R01-GM088403). The nanopore drilling was performed at the Electron Microscopy Facility of the Cornell Center for Materials Research (CCMR) with support from the National Science Foundation - Materials Research Science and Engineering Centers (MRSEC) program (DMR 0520404). The preparation of the silicon nitride membranes was performed at the Cornell NanoScale Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS-0335765).

1. Manufacture of solid-state nanopores in silicon nitride membranes
<ol>
 <li>Bring the FEI Tecnai F20 S/TEM to an acceleration voltage of 200 kV. If using a different S/TEM, the acceleration voltage should be greater than or equal to 200 kV 9</li>
 <li>Load a 20 nm thick SPI silicon nitride window grid into the TEM sample holder and clean with Oxygen plasma for 30 seconds to remove any contaminants from the holder.</li>
 <li>Load the sample into the S/TEM and allow for the vacuum to pump down. Once the S/TEM has pumped down to vacuum, find the nitride window in bright-field TEM mode by looking for a bright square on the Ronchigram. Make sure the TEM is aligned properly, then align and focus the sample.</li>
 <li>Switch the S/TEM into STEM mode. Use the HAADF (or equivalent) detector to image the sample and make sure it is properly aligned.</li>
 <li>Set the Monochromator to a low value. A lower value of the Monochromator allows for a higher current of electrons available for drilling. If the S/TEM does not have a Monochromator, ignore this step.</li>
 <li>Select an appropriate spot size for drilling the nanopore. This corresponds to the diameter of the electron probe. A 3 nm probe works well for drilling 5 nm diameter pores. A larger probe (5 nm) will drill more quickly and create a larger pore. A smaller probe (1 nm) will take longer to drill and create a smaller pore.</li>
 <li>Adjust the STEM alignments, if necessary.</li>
 <li>Focus the nitride membrane and bring it to a magnification of x1.3M. Fine focusing should be done by monitoring the Ronchigram.</li>
 <li>If there is any movement of the sample, then wait for the sample to stabilize. This can take up to an hour. Blank the electron beam while waiting so as not to damage the nitride.</li>
 <li>Place the electron probe on the sample and begin drilling.</li>
 <li>Periodically check the sample by scanning with the HAADF detector, both to make sure the nanopore is being drilled (it will look like a dark spot) and to check that there is no movement of the sample. If the sample drifts slightly, adjust the position of the electron probe to be over the nanopore.</li>
 <li>Watch the Ronchigram to identify when the pore has formed. When in focus, the nitride film has a shimmering appearance. This will disappear when the nanopore forms. Putting the Ronchigram slightly out of focus allows one to see a Fresnel fringe associated with the nanopore.</li>
 <li>Take an image of the nanopore with the HAADF.</li>
 <li>Do an intensity profile of the nanopore image. The diameter of the nanopore can be estimated by the darkest region of the profile.</li>
 <li>Enlargement of the nanopore may be performed by moving the electron probe along the edges of the pore.</li>
 <li>Once the nanopore is of the desired diameter, put the S/TEM into TEM mode.</li>
 <li>Bring the Monochromator to its standard setting and image the nanopore using bright-field TEM to confirm the size (Fig. 1).</li>
</ol>
2. Wetting of the solid-state nanopore
<ol>
 <li>De-gas de-ionized water containing 1 M KCl, 10 mM potassium phosphate, pH 7.4. This can be done by putting the solutions under vacuum and placing them in a bath sonicator for 20 minutes.</li>
 <li>Place the nanopore containing silicon nitride chip into a 10 ml Pyrex beaker. Take care not to break the nitride window as it is very delicate. Place the beaker on a hotplate in a fume hood and set to 100&deg;C.</li>
 <li>Clean the nanopore chip with piranha solution using great care. First add 3 ml sulfuric acid to the container using a glass pipette. Next, carefully add 1 ml hydrogen peroxide to the sulfuric acid to make piranha solution14. Please take all precautions.</li>
 <li>Allow the nanopore chip to soak in piranha solution for 10 minutes.</li>
 <li>Remove the piranha solution from the beaker using a glass pipette. Place it into a proper storage receptacle.</li>
 <li>Fill the beaker with the de-gassed, de-ionized water using a clean glass pipette. Empty beaker of water and repeat at least 5 times.</li>
 <li>Remove the nanopore chip with clean tweezers and dry it by light suction</li>
 <li>Immediately, seal the nanopore chip into the chamber (Fig. 2a, 2b). The chip may be secured in the chamber by O-rings or silicone sealant.</li>
 <li>Fill the chamber with KCl solution</li>
 <li>Connect the Ag/AgCl electrodes to the chamber (Fig. 2b). Electrodes may be made by soaking silver wire in bleach overnight.</li>
 <li>Apply a transmembrane potential across the electrodes and monitor the current response using an Axon 200B patch-clamp amplifier in the voltage-clamp mode.</li>
 <li>Construct an I/V (current-voltage) curve from the measurements. The I/V curve should be highly linear when using 1 M KCl (Fig. 3). The conductance of the nanopore should correspond to its diameter. If the nanopore does not display a linear I/V curve, the conductance is too low, or the open current of the nanopore is not stable (Fig. 4a), it means that the nanopore is not properly wetted and piranha washing should be repeated.</li>
</ol>
3. Monitoring protein adsorption
<ol>
 <li>It is critically important to perform control experiments with a single-nanopore membrane or parallel array of nanopores (see below) to monitor the current with the buffer conditions lacking the protein sample. We recommend the following two steps: (i) the use of a high purity-level buffer (purity &gt; 99.9%; chromatography-grade level), and (ii) the use of a buffer whose interaction with the solid-state nanopore does not produce single-channel events. In addition, we recommend obtaining a high-purity protein sample using standard protein chemistry procedures. The purity of the protein sample should be checked by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel analysis and high-resolution mass spectrometry 15.</li>
 <li>Add a purified sample of the protein to be studied into the grounded bath of the chamber. Allow time for the sample to diffuse throughout the bath. Typical concentrations of protein that can be measured are in the tens of nM to &mu;M range 7,15-21.</li>
 <li>Apply a transmembrane potential voltage bias with polarity opposite to that of the total charge of the protein being studied. For example, BSA has an effective net negative charge at pH 7.4, and therefore a positive voltage bias should be applied. The applied voltage creates potential gradient within the nanopore interior and proteins of opposite charges will be driven by opposite forces. Only voltage polarities that drive the proteins into the nanopore will create measurable signals.</li>
 <li>An applied potential of magnitude of 200 mV should be large enough to observe most protein analytes. Events will appear as transient current deflections from the baseline (Fig. 4b). If no signal is observed, increase the concentration of the protein in the chamber. Higher voltages improve the signal to noise ratio. Nitride nanopores can withstand several volts.</li>
 <li>Protein adsorptions will appear as long-lived changes in the baseline current of the nanopore (Fig. 4c).</li>
</ol>
4. Possibilities for functionalization of the nanopores
There exist several methods for applying functional groups to silicon nitride 22,23. Most nitride films have a thin oxide layer on the outside and exposure to ozone can create an additional oxide layer, if necessary. Different organosilanes can be used and self-assembled onto such layers. Of particular interest is aminosilane, which self-assembles and can be modified further (i.e., carboxylic acid and aldehydes), allowing for the inspection of a range of organic surfaces. After a nanopore has been washed with piranha and secured in the chamber, amine can be added directly to the chamber bath with a supporting electrolyte of 0.5 M TBACl (tetrabutylammonium) and anhydrous MeOH (methanol) used as a solvent. Monolayer formation can be monitored by the application of a 400 mV voltage bias and the measurement of the current drop 23.
5. Determination of the apparent first-order reaction rate constant and the Langmuir adsorption constant using parallel nanopore arrays
5.1 The apparent first-order reaction rate constants for absorption and desorption
We emphasize that the positive aspect of this methodology is its ability to observe individual adsorptions at the single-molecule level. The single-molecule measurements of protein adsorption onto an inorganic surface can be scaled up by employing parallel arrays of solid-state nanopores. The parallel array of nanopores is necessary because of the need to assay many pores to get reliable statistics. To this end, the use of a nanopore array would allow the monitoring of individual adsorptions as well as the measurement of multiple events for further analysis. An array of nanopores in silicon nitride may be formed by the above protocol simply by drilling multiple holes in the sample. Each nanopore should be of similar size. Arrays of nanopores of 6x6 or 7x7 should be adequate. Upon addition of protein analyte in the chamber, the current will decay in an exponential manner with a following expression: 
 
It = I&infin; - (I&infin; - I0)exp(-k't)
Here, It, denotes the current at an experimental time t. I0 is the original current passing through the nanopore array. I&infin; indicates the current at saturation level (i.e., infinity). k' is the apparent first-order reaction rate constant, which can be determined from the fit of the experimental curve. A greater k' can be interpreted as a faster adsorption rate. The ratio I&infin;/I0, also called the normalized saturation current, is a dimensionless number between 0 and 1. This parameter is a measure of the occupancy by the adsorbed protein analytes. Therefore, each distinct experimental condition should be associated with two specific output parameters I&infin;/I0 and k'.
It should be noted that the apparent first-order adsorption rate constants and normalized currents are impacted by the effective time spent by proteins within the nanopores. This time is dependent on the concentration of protein analytes in bulk aqueous phase 24. Therefore, additional correction of these numbers needs to be implemented based upon the effective time spent by the proteins within the nanopore interior. We suggest measuring the frequency of fast (translocation) events and multiplying it by the average dwell time of such events. This will give the average time of proteins spent within the nanopore interior per unit time.
The rate constant of desorption can be determined using a parallel array of nanopores as well. As soon as the current level reaches saturation (I&infin;), the voltage should be reversed, so no more proteins are trapped into the nanopores. Desorption of individual proteins will be accompanied by the alteration of the recorded current towards greater values. The rate constant will be then extracted from the rise in the current level.
5.2 The Langmuir adsorption constant
The absorption of protein analytes onto the inorganic surfaces of the nanopores is dependent on the protein concentration in aqueous phase. This phenomenon has already been observed at the single-molecule level 13. If &theta; represents the normalized saturation current (I&infin; / I0), then a typical Langmuir isotherm equation is given by the following expression:
<img alt="Figure 5.2" src="/files/ftp_upload/3560/3560fig5_2.jpg" />
where C is the protein concentration in the aqueous phase. &alpha; is the Langmuir adsorption constant. This constant increases with an increase in the binding energy of adsorption and with a decrease in temperature. The collection of the &theta; data points will employ measurements with parallel arrays carried out at various protein concentrations in aqueous phase. Data should be analyzed in combination with other techniques to verify the magnitude of the fitted Langmuir adsorption constant. In addition, full-atomistic molecular dynamics simulations 25,26 might be also used to help the interpretations of the obtained experimental data.
6. Representative Results
Typical results for solid-state nanopores will be as follows. The open pore current should be highly stable, as seen in Fig. 4a. The I/V characteristics of the nanopore should be highly linear in 1 M KCl, 10 potassium phosphate, pH 7.4, as seen in Fig. 2. The slope of a linear fit to the I/V curve will provide the unitary conductance of the nanopore. The conductance has a direct relationship to the nanopore diameter and should match the equation: <img alt="Figure equation" src="/files/ftp_upload/3560/3560figeqn.jpg" />, where G is the conductance of the nanopore, d is its diameter, l is its length, and &sigma; is the conductivity of the solution in the chamber 14. This value should match to within 30%. If it is too small, your pore is likely not wetted. With addition of protein, rapid events should ensue, as seen in Fig. 4b. Protein adsorption is a long-lived current drop as displayed in Fig. 4c. Some proteins are highly labile and undergo structural transformation within the pore interior. In this case, the long-lived voltage drop will be accompanied by rapid fluctuations.
<img alt="Figure 1" src="/files/ftp_upload/3560/3560fig1.jpg" /> 
Figure 1. Solid-state nanopore fabricated using a Tecnai F20 S/TEM. The pore was drilled in STEM mode to a diameter of 20 nm. The image was taken in bright-field TEM mode. Nitride was 30 nm thick.
<img alt="Figure 2" src="/files/ftp_upload/3560/3560fig2.jpg" /> 
Figure 2. Chamber used for housing a single solid-state nanopore in resistive-pulse measurements. A) Chamber and a silicon chip with free-standing nitride window (TEM grid). The nanopore is drilled into the nitride before loading. Red O-rings are used to form a good seal about the chip, which separates two baths of ionic solution. B) Schematic of the chamber showing placement of solution baths and electrodes with respect to the nanopore.
<img alt="Figure 3" src="/files/ftp_upload/3560/3560fig3.jpg" /> 
Figure 3. Typical I/V traces for solid-state nanopores of different diameters. Single-channel electrical traces were taken in 1 M KCl, 20 mM Tris, pH 8.5. Nanopores were drilled in 30 nm thick silicon nitride. Note nitride thickness will affect the unitary conductance of the nanopore.
<img alt="Figure 4" src="/files/ftp_upload/3560/3560fig4.jpg" /> 
Figure 4. Measured single-channel current traces and the detection of protein adsorption. A) A single-channel electrical trace showing the open current of a 10 nm diameter nanopore. B) Current deflections represent partitioning of bovine serum albumin (BSA) into the interior of the nanopore. C) Adsorption of BSA onto the nitride surface. All traces are from the same nanopore in 1 M KCl, 10 mM potassium phosphate, pH 7.4. The applied transmembrane potential was +40 mV and BSA was added to the grounded chamber bath at a concentration of 120 nM.

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H., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+electron+microscopy+'windows'+for+nanofabricated+structures.">Transmission electron microscopy 'windows' for nanofabricated structures.</a> Nanotechnology. 15, 1175-1181 (2004).</li><li>Giannoulis, C. S., Desai, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+proteins+and+fibroblasts+on+thin+inorganic+films.">Characterization of proteins and fibroblasts on thin inorganic films.</a> J. Mater. Sci: Mater. Med. 13, 75-80 (2002).</li><li>Gustavsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modifications+of+silicon+nitride+for+cellular+biosensors+applications.">Surface modifications of silicon nitride for cellular biosensors applications.</a> J. Mater. Sci: Mater. Med. 19, 1839-1850 (2008).</li><li>Shirshov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+response+of+planar+polarization+interferometer+to+molecular+layer+formation:+fibrinogen+adsorption+on+silicon+nitride+surface.">Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface.</a> Biosens. Bioelectron. 16, 381-390 (2001).</li><li>Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+layer+deposition+to+fine-tune+the+surface+properties+and+diamters+of+fabricated+nanopores.">Atomic layer deposition to fine-tune the surface properties and diamters of fabricated nanopores.</a> Nano. Lett. 4, 1333-1337 (2004).</li><li>Siwy, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosensors+based+on+biofunctionalized+conical+gold+nanotubes.">Protein biosensors based on biofunctionalized conical gold nanotubes.</a> J. Am. Chem. Soc. 127, 5000-5001 (2005).</li><li>Ding, S., Gao, C., Gu, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+Single+Molecules+of+Immunoglobulin+and+Ricin+with+an+Aptamer-Encoded+Glass+Nanopore.">Capturing Single Molecules of Immunoglobulin and Ricin with an Aptamer-Encoded Glass Nanopore.</a> Anal. Chem. , (2009).</li><li>Kim, M. -. J., Wanunu, M., Bell, C. D., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Fabrication+of+Uniform+Size+Nanopores+and+Nanopore+Arrays+for+Parallel+DNA+Analysis.">Rapid Fabrication of Uniform Size Nanopores and Nanopore Arrays for Parallel DNA Analysis.</a> Adv. Mater. 18, 3149-3153 (2006).</li><li>Bezrukov, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+as+molecular+Coulter+counters+to+probe+metabolite+transport.">Ion channels as molecular Coulter counters to probe metabolite transport.</a> J. Membr. Biol. 174, 1-13 (2000).</li></ol>

We have nothing to disclose.

Spontaneous adsorption of proteins onto solid-state surfaces 27-29 is fundamentally important in a number of areas, such as biochip applications and design of a new class of functional hybrid biomaterials. Previous studies have shown that proteins adsorbed to solid-state surfaces do not show lateral mobility or significant desorption rates, and therefore protein adsorption is generally considered an irreversible and nonspecific process 30-32. Protein adsorption onto solid state surfaces is thought t...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tecnai F20 S/TEM</td>
 <td>FEI</td>
 <td>&nbsp;</td>
 <td>S/TEM requires acceleration voltage &ge;200kV and field-emission source. </td>
 </tr>
 <tr>
 <td>20 nm thick silicon nitride membrane window for TEM</td>
 <td>SPI</td>
 <td>4163SN-BA </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Axopatch 200B patch-clamp amplifier</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Digidata 1440A</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pCLAMP 10 software</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>Electrophysiology Data Acquisition and Analysis Software</td>
 </tr>
 <tr>
 <td>Sulfuric Acid</td>
 <td>Fisher Scientific</td>
 <td>A300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hydrogen peroxide</td>
 <td>Fisher Scientific</td>
 <td>H325</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>silicone O-rings</td>
 <td>McMaster-Carr</td>
 <td>003 S70</td>
 <td>Alternatively use PDMS </td>
 </tr>
 <tr>
 <td>silver wire</td>
 <td>Sigma-Aldrich</td>
 <td>348759</td>
 <td>For electrodes</td>
 </tr>
 <tr>
 <td>SPC Technology, D sub contact, pin</td>
 <td>Newark</td>
 <td>9K4978 </td>
 <td>For electrodes</td>
 </tr>
 <tr>
 <td>potassium chloride</td>
 <td>Sigma</td>
 <td>P9541</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>potassium phosphate dibasic</td>
 <td>Sigma</td>
 <td>P2222</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>potassium phosphate monobasic</td>
 <td>Sigma</td>
 <td>P5379</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PDMS</td>
 <td>Dow Corning</td>
 <td>&nbsp;</td>
 <td>Sylgar 184 Elastomer set. For making chamber.</td>
 </tr>
 <tr>
 <td>Kwik-Cast Sealant</td>
 <td>World Precision Instruments</td>
 <td>KWIK-CAST</td>
 <td>Fast acting silicone sealant</td>
 </tr>
 <tr>
 <td>hot plate</td>
 <td>Fisher Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday cage</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
</table>

monitoring protein adsorption with solid-state nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

Watch this Scientific Journal Video about Monitoring Protein Adsorption with Solid-state Nanopores at JoVE.com

Monitoring Protein Adsorption with Solid-state Nanopores

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

monitoring-protein-adsorption-with-solid-state-nanopores

Research

JoVE Journal

Bioengineering

13.5K Views.  Syracuse University. Метод с использованием полупроводниковых нанопор для контроля неспецифической адсорбции белков на поверхности неорганические описывается. Метод использует резистивный импульса принципу, что позволяет адсорбции быть проверены в режиме реального времени и...

Video: Monitoring Protein Adsorption with Solid-state Nanopores

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Construction of a Wireless-Enabled Endoscopically Implantable Sensor for pH Monitoring with Zero-Bias Schottky Diode-based Receiver

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or non-acidic refluxate. This paper describes a method for the development, manufacturing, and implantation of a miniature wireless-enabled pH sensor. The sensor is designed to be implanted endoscopically with a single hemostatic clip. A fully passive rectenna-based receiver based on a zero-bias Schottky diode is also constructed and tested. To construct the device, a two-layer printed circuit board and off-the-shelf components were used. A miniature microcontroller with integrated analog peripherals is used as an analog front end for the ion-sensitive field-effect transistor (ISFET) sensor and to generate a digital signal which is transmitted with an amplitude shift keying transmitter chip. The device is powered by two primary alkaline cells. The implantable device has a total volume of 0.6 cm3 and a weight of 1.2 grams, and its performance was verified in an ex vivo model (porcine esophagus and stomach). Next, a small footprint passive rectenna-based receiver which can be easily integrated either into an external receiver or the implantable neurostimulator, was constructed and proven to receive the RF signal from the implant when in proximity (20 cm) to it. The small size of the sensor provides continuous pH monitoring with minimal obstruction of the esophagus. The sensor could be used in routine clinical practice for 24/96 h esophageal pH monitoring without the need to insert a nasal catheter. The &#34;zero-power&#34; nature of the receiver also enables the use of the sensor for automatic in-vivo calibration of miniature lower esophageal sphincter neurostimulation devices. An active sensor-based control enables the development of advanced algorithms to minimize the used energy to achieve a desirable clinical outcome. One of the examples of such an algorithm would be a closed-loop system for on-demand neurostimulation therapy of gastroesophageal reflux disease (GERD).

The manuscript presents a miniature implantable pH sensor with ASK modulated wireless output together with a fully passive receiver circuit based on zero-bias Schottky diodes. This solution can be used as a basis in the development of in vivo calibrated electrostimulation therapy devices and for ambulatory pH monitoring.

Ambulatory pH monitoring of pathological reflux is an opportunity to observe the relationship between symptoms and exposure of the esophagus to acidic or ...