This work was funded by Alliance of NanoHealth Pre-center Award (W81XWH-11-2-0168) and Texas Center for Cancer Nanomedicine (1U54CA151668-01).

1. Chip Fabrication
<ol>
	<li>Create the coating solution for the chip by starting with the hydrolyzed silicate precursor solution. Mix 14 mL of tetraethylorthosilicate (TEOS) with 17 mL of ethanol, 6.5 mL of deionized water and 0.5 mL of 6M HCl under strong stirring (1200 rpm) using a stir hot plate. Heat this solution at 80 &deg;C for 2 hours, keeping the stirring constant.</li>
	<li>Prepare polymer solutions by adding the desired tri-block coploymer (pluronic F127, L121 and P123) to 10 mL of ethanol at room temperature with strong stirring. Complete the mixture by adding 7.5 ml of the silicate solution (from step 1.1) into the tri-block copolymer solution followed by 2 hours of strong stirring at room temperature. This represents the final coating solution.</li>
	<li>Apply 1 mL of coating solution to a 4 inch silicon wafer by spin-coating at a rate of 1500 rpm for 20 seconds. Then heat at 80 &deg;C for 12 hours.</li>
	<li>Heat the films to remove organic surfactant by raising temperature 1 &deg;C per minute to 425 &deg;C, and then bake for 5 hours.</li>
	<li>Pretreat the mesoporous silica (MPS) chip surface with oxygen plasma ashing (Plasma Asher - March Plasma System). (O2 Flow rate: 80 sccm, power: 300 W, time: 10 minutes).</li>
	<li>Optional surface chemical modification: silanate chips in 3% organosilane in a Methanol:DI water (19:1) solution for 72 hours at room temperature in an N2 glove box. Rinse sequentially with Methanol and DI water. Cure chips at 110 &deg;C for 15 minutes in a fan-operated oven.</li>
</ol>
2. Sample Pre-treatment
<ol>
	<li>Add TFA and ACN to each serum sample such that the final concentrations are 0.01% TFA and 5% ACN. Vortex to mix.</li>
	<li>Shake these samples on a table vortex shaker at room temperature for 30 minutes.</li>
</ol>
3. Serum Fractionation
<ol>
	<li>Pre-bake chips over night in an oven at 160 &deg;C. Alternatively, store the chips in a desiccator until ready to use to prevent hydration of the surface by ambient water in air.</li>
	<li>Use compressed air to dust off any particles that may be on the surface of the chip.</li>
	<li>Cut prefabricated, purchased CultureWell chambered coverglass to include the desired number of wells. Clean coverglass with 100% ethanol and then place on MPS chip surface. Press coverglass down with forceps to ensure a complete seal with chip.</li>
	<li>Pipette 10 &mu;L of serum sample into each 3 mm well. Incubate for 30 minutes in a humidified chamber at room temperature.</li>
	<li>Pipette up serum from wells and discard. Pipette 10 &mu;L of deionized water to each well to wash away larger proteins. Repeat 4 times.</li>
	<li>Pipette 5 &mu;L elution buffer (0.1%TFA + 50% ACN) to each sample well. Pipette up and down 30 times while moving the pipette tip around in the well to mix the elution buffer. (Only apply elution buffer to 1-2 samples at a time to prevent elution buffer from evaporating so quickly).</li>
	<li>After mixing, pipette all the elution buffer and place in a microcentrifuge tube until ready to perform MALDI-TOF analysis.</li>
	<li>In order to emulate the complexity of a biological sample and to evaluate the enrichment effect in harvesting low molecular weight species with our on-chip fractionation system, we selected and assembled a standard mixture of proteins and peptides counting twenty-six different species with a broad range of molecular weights (900-66 500 Da) and pIs (4.0-10.2) and concentrations (0.5-8 pmol/&mu;l ) (see protein list in Table 1).</li>
</ol>
4. MALDI-TOF Analysis of Peptides
<ol>
	<li>Spot 0.5 &mu;L of sample to MALDI target plate and allow to dry.</li>
	<li>Spot 0.5 &mu;L matrix (&alpha;-cyano-4-hydroxycinnamic acid (CHCA), 5 g/L) or saturated solution of trans-3,5-dimethoxy-4-hydroxycinnamic acid (SA) in 50% acetonitrile containing 0.1% TFA and allow to co-crystallization.</li>
	<li>Spot 0.5 &mu;L of calibration solution to each calibration spot and allow to dry.</li>
	<li>Insert target plate into MALDI-TOF mass spectrometer. The machine should be set to positive reflector mode with a laser intensity of 4200 and 3000 shots per sample. The selected mass range should be 800 to 5000 Da with a target mass of 2000 Da.</li>
	<li>Perform same MALDI-TOF analysis in linear mode but change the mass range to 900 to 10,000 Da or 3000 to 70,000 Da and a target mass of 5000 Da.</li>
</ol>
5. Data Analysis
<ol>
	<li>The raw spectra were processed with the ConvertPeakList software and the data was exported to SpecAlign software for preprocessing. All spectra were aligned using the PAFFT correlation method and intensities were normalized to total ion current (TIC) in each corresponding spectrum. All spectra were smoothed and de-noised with factor of 4 and 0.5 respectively. Peaks were detected with a baseline of 0.5, mass window of 21 and height ratio 1.5, negative values were removed before analysis.</li>
	<li>Hierarchical clustering was performed using Cluster 3.0 and visualized with MapleTree software. MALDI MS Data (m/z peak intensities) was log-transformed, normalized, and median centered. Pearson correlation was used to calculate the distance between the samples, and complete linkage clustering was performed.</li>
	<li>An independent Student t-test was used for comparison between groups (n = 2 groups) for each detected MS peak prior to unsupervised hierarchical clustering analysis. A P-value of 0.02 or lower was considered significant to select differentially harvested peptides and proteins among the different mesoporous proteomic chips (Large pores vs. Small pores).</li>
</ol>
6. Representative Results
As shown in Figure 1, in this study we fabricated a series of mesoporous silica thin films with a variety of nanotextures and comprehensively explored their use in selective capturing and enriching LMW peptides and proteins from human serum. Figure 2a and b show the MS spectra of the unprocessed serum sample for peptides in the range of 900 to 10,000 Da and for proteins in the range of 3,000 ~ 70,000 Da respectively. These spectra illustrate the signal suppression in the LMW region due to the presence of well ionized, highly abundant, high molecular weight (HMW) proteins such as Albumin. Figure 2c and d depict the MS spectra of the serum sample after fractionation by the MPS L121 (pore size, 6 nm). The majority of the large molecules have been depleted, resulting in a significant enrichment of the LMW components. As a control, the same serum sample was applied onto a nonporous pure silica surface to evaluate the specificity of MPS thin films for LMWP recovery. As can be seen in Figure 2e and f, there was no significant harvesting of peptides or proteins from the nonporous silica. Thus it can be concluded that it was the mesoporous architecture and not the silica surface affinity that constitutes the predominate factor in the enrichment of LMWP.
Precisely controlled variations in pore size can be achieved through the use of copolymers with differing hydrophobic block lengths. By using the designed proteins and peptides mixture, the effect of pore size on the LMW peptide and protein recovery efficacy was investigated using MPS thin films prepared from four Pluronic surfactants (F127, P123, L121, and L121 plus swelling agent) with different volume ratios of the hydrophilic and hydrophobic components to form pore sizes of 3.7 nm, 5.2 nm, 7.4 nm, and 9.0 nm respectively. This range of pore sizes led to the recovery of a different repertoire of peptides and proteins from the same serum sample via size and shape exclusion (Figure 3). The protein spectra at high molecular weight demonstrate the molecular cut-off of each type of chip. In addition to the size-dependant depletion of HMW proteins, the on-chip fractionation of the standards solution displays a differential and selective enrichment of LMW species associated with the pore sizes. The two-way hierarchical clustering presented in Figure 3b shows the LMW standards enrichment pattern obtained with the different MSC. Even if all the peptides are below the molecular cut-off of the chips, there is a positive correlation between the pore sizes and the molecular weight of the trapped species. The MSC with large pores, up to 9 nm, preferentially harvest bigger peptides, while smaller peptides are recovered more efficiently by the chips with smaller pores. The structural transformation of the mesoporous arrangement was carried out by tuning the concentration of the template polymer. Increasing the concentration of the template polymer resulted in a reduced interfacial curvature between the phases of the water, the copolymer, and the silicate, consequently initiating the interrelated progression from a spherical to a cylindrical structure. Pluronic F127, with its high molecular weight, possesses this high degree of structural periodicity. By increasing the concentration of F127 in starting solution, different MPS thin film periodic nanostructures can be obtained from 3D nanostructure to 2D nanostructure. The 3D cubic and honeycomb hexagonal nanostructures, possessing more desirable nanopore interconnectivity and more accessible nanopore morphology, exhibit superior performance in selectively enriching LMW peptides than the 2D hexagonal structure, even though they share similar pore size distributions and the same molecular cut-off for serum fractionation (Figure 4). We also streamlined the conjugation of organo-silane on MPS chips by introducing oxygen plasma ashing to pretreat the chip surface. In order to qualitatively study the electrostatic effect on selective on-chip enrichment, we use the proteins and peptides mixture. MS analysis of the proteomic standards solution fractionated on MPS chips prepared with L121 and conjugated with the chemical functional groups is presented in Figure 5. The positively charged and negatively charged the peptides and LMW proteins are captured on the anionic and the cationic chips respectively. The quantitative comparison of multiple MPS chips in recovering the peptides with positive net charge is displayed in Figure 5a. The chips with negative charge and the chips without any modification (with a minor negative charge originally) exhibit significantly higher enrichment for those peptides than the chips modified with APTES (-NH2). Conversely, the positively charged MPS chips possess exceptional capability to recover those peptides with negative net charge, as demonstrated in Figure 5b. While &alpha;-Endorphin does not show a significant change due to its PI around 6. 
<img alt="Figure 1" src="/files/ftp_upload/3876/3876fig1.jpg" /> 
Figure 1. Principle of MPS chips fractionation and LMW enrichment. After sample spotting on the surface, LMW proteins and peptides are trapped into the pores while the larger species remained outside the pores and are removed during the washing steps. The enriched fractions are then eluted and analyzed by MALDI.
<img alt="Figure 2" src="/files/ftp_upload/3876/3876fig2.jpg" /> 
Figure 2. Peptide enrichment using the mesoporous silica thin film chips. MALDI MS profiles in both the low mass range (900 to 10 000 Da) and the high mass range (3000 to 70 000 Da) before (a,b) and after (c,d) serum processing on the mesoporous silica thin films (L121, 6 nm). The molecular recovery is significantly reduced when using blank nonporous silica surfaces (e,f).
<img alt="Figure 3" src="/files/ftp_upload/3876/3876fig3.jpg" /> 
Figure 3. Molecular cut-off and size-dependent enrichment of the MPS chips. (a) Magnified view of the MALDI spectra demonstrating the characteristic molecular cut-off of each MPS chips correlating to the pore size. (b) Two-way hierarchical clustering of the peptide mix features among the different chips. The intensity of the red or yellow color indicates the relative peptide concentration. Larger pores enhanced the harvesting of larger peptides (from 3600 to 8500 Da), while the small peptides (from 900 to 3500 Da) were preferentially recovered from the chips with smaller pores.
<img alt="Figure 4" src="/files/ftp_upload/3876/3876fig4.jpg" /> 
Figure 4. Physical characterizations of MPS thin films and selective recovery with different nanostructures. XRD patterns (a, b, c), TEM (inset a, b, c), Pluronic F127 at different concentrations in the precursor solution: 4.0&times;10-3 M (a), 6.0&times;10-3 M (b), and 8.0&times;10-2 M (c). (d) Bar graph of the intensity of detection illustrating the selective peptides recovery on 3D cubic and 3D hexagonal F127 proteomic chips (Cub and Hex, respectively). The different structural modifications present a selective enrichment.
<img alt="Figure 5" src="/files/ftp_upload/3876/3876fig5.jpg" /> 
Figure 5. Charge-specific recovery for the chips with different surface functions. Bar graph of the MS intensity of detection of selectively captured peptides on the functionalized chips. According to their iso-electric point, the peptides are positively or negatively charged at pH 7.0. (a) Positive peptides ((1) des-Arg1-Bradykinin, (2) Bradykinin, (3) substance P-amide, (4) Neurotensin, (5) ACTH(1-17), (6) ACTH(7-38)) are specifically enriched on the negatively charged surfaces. (b) Negative peptides ((7) Glu1-fibrinopeptide B, (8) &alpha;-endorphin, (9) ACTH(18-39), (10) insulin, (11) EGF, (12) insulin-like GFII) are specifically enriched on the positively charged surfaces. Please <a href="https://www.jove.com/files/ftp_upload/3876/3876fig5_lg.jpg">click here</a> to see a larger version of this figure.
<img alt="Figure 6" src="/files/ftp_upload/3876/3876fig6.jpg" /> 
Figure 6. On-chip stabilization of fractionated serum. (a) Representative MALDI profiles of LMW peptides and proteins eluted immediately after serum fractionation (top) or after 3 wk of on-chip storage at room temperature (bottom). (b) From top to bottom: Linear regression analysis of average intensities of detected MS peaks in each replicate compared to replicate 1 for freshly fractionated serum and fractionated serum after 3wk MPS chips storage at room temperature. Equation, CV and coefficient of determination (R2) are indicated. Please <a href="https://www.jove.com/files/ftp_upload/3876/3876fig6_lg.jpg">click here</a> to see a larger version of this figure.

<ol>
	<li>Wulfkuhle, J. D., Liotta, L. A., Petricoin, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+applications+for+the+early+detection+of+cancer.">Proteomic applications for the early detection of cancer.</a> Nat. Rev. Cancer. 3, 267-275 (2003).</li><li>Etzioni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+case+for+early+detection.">The case for early detection.</a> Nat. Rev. Cancer. 3, 243-252 (2003).</li><li>Hanash, S. M., Pitteri, S. J., Faca, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mining+the+plasma+proteome+for+cancer+biomarkers.">Mining the plasma proteome for cancer biomarkers.</a> Nature. 452, 571-579 (2008).</li><li>Liotta, L. A., Ferrari, M., Petricoin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+proteomics:+written+in+blood.">Clinical proteomics: written in blood.</a> Nature. 425, 905-905 (2003).</li><li>Petricoin, E. F., Belluco, C., Araujo, R. P., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+peptidome:+a+higher+dimension+of+information+content+for+cancer+biomarker+discovery.">The blood peptidome: a higher dimension of information content for cancer biomarker discovery.</a> Nat. Rev. Cancer. 6, 961-967 (2006).</li><li>Anderson, N. L., Anderson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+plasma+proteome:+history,+character,+and+diagnostic+prospects.">The human plasma proteome: history, character, and diagnostic prospects.</a> Mol. Cell Proteomics. 1, 845-867 (2002).</li><li>Gorg, A., Weiss, W., Dunn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+two-dimensional+electrophoresis+technology+for+proteomics.">Current two-dimensional electrophoresis technology for proteomics.</a> Proteomics. 4, 3665-3685 (2004).</li><li>Liu, H., Sadygov, R. G., Yates, J. R., 3rd, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+random+sampling+and+estimation+of+relative+protein+abundance+in+shotgun+proteomics.">A model for random sampling and estimation of relative protein abundance in shotgun proteomics.</a> Anal. Chem. 76, 4193-4201 (2004).</li><li>Rabilloud, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+gel+electrophoresis+in+proteomics:+old,+old+fashioned,+but+it+still+climbs+up+the+mountains.">Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains.</a> Proteomics. 2, 3-10 (2002).</li><li>Hu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+of+the+nanotexture+of+mesoporous+silica+films+and+their+functionalized+derivatives+for+selectively+harvesting+low+molecular+weight+protein.">Tailoring of the nanotexture of mesoporous silica films and their functionalized derivatives for selectively harvesting low molecular weight protein.</a> ACS Nano. 4, 439-451 (2010).</li><li>Bouamrani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoporous+silica+chips+for+selective+enrichment+and+stabilization+of+low+molecular+weight+proteome.">Mesoporous silica chips for selective enrichment and stabilization of low molecular weight proteome.</a> Proteomics. 10, 496-505 (2010).</li></ol>

We have nothing to disclose.

Evidence is mounting that the low molecular-weight region of the circulatory proteome is a rich source of diagnostic biomarkers for the early detection of disease. In this technology, we presented a series of mesoporous silica chips with different pore sizes, pore structures and modifications to selectively enrich peptides and low molecular weight proteins. To evaluate protein stability, the MPS chips were incubated with human serum, dried after washing, and stored for 3wk at room temperature. The protein/peptide patterns obtained were comparable with those of freshly fractionated serum (Figure 6a), as confirmed by the results of the statistical analysis showed in Figure 6b. The variability of the peak signals measured by the average CV was estimated at 12.7% for crude serum and at 14.2% for the fractionated samples. The marginal variations could be due to the internal variability of the MALDI instrument and suggested that the on-chip pretreatment and storage did not induce any significant alteration of the MS protein profiles. The same experiment has been performed on non porous silicon. The dried serum recovered after storage on silicon surface was fractionated on the MPS chips before MALDI analysis. The poor MS profile obtained demonstrated the stabilization advantage of the mesoporous surface. In analogy with previously postulated mechanisms, we hypothesize that the LMW species trapped inside the nanopores were preserved from degradation through the size exclusion of proteases, or by steric inhibition of their proteolytic activity in the confined space of the nanopores. The MPS chip based method could be a powerful tool in the peptide and LMW protein profiling of complex biological fluids study. The MPS chips are inexpensive to manufacture and allow for scaled up production to attain the simultaneous processing of a large number of samples, providing advantageous features for exploratory screening and biomarker discovery.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tetraethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>131903</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Brewer Science</td>
			<td>Cee 200X</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plasma Asher</td>
			<td>Nordson March</td>
			<td>AP-600</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectroscopic Ellipsometer</td>
			<td>J. A. Woollam Co.</td>
			<td>M-2000DI</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MALDI-TOF</td>
			<td>Applied Biosystems</td>
			<td>Voyager-DE-STR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&alpha;-cyano-4-hydroxycinnamic acid</td>
			<td>Sigma-Aldrich Co.</td>
			<td>C8982</td>
			<td>Matrix for MALDI-TOF</td>
		</tr>
		<tr>
			<td>trans-3,5-dimethoxy-4-hydroxycinnamic acid</td>
			<td>Sigma-Aldrich Co.</td>
			<td>85429</td>
			<td>Matrix for MALDI-TOF</td>
		</tr>
		<tr>
			<td>Stir hot plate</td>
			<td>Thermo Scientific</td>
			<td>11-475-30Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CultureWell chambered coverglass</td>
			<td>Sigma-Aldrich</td>
			<td>GBL103350</td>
			<td>3 mm diam. &times;1 mm depth, 3-10 &mu;L, sterile</td>
		</tr>
		<tr>
			<td>Pluronic F 127</td>
			<td>BASF</td>
			<td>&nbsp;</td>
			<td>PEO106-PPO70-PEO106</td>
		</tr>
		<tr>
			<td>Pluronic L121</td>
			<td>BASF</td>
			<td>&nbsp;</td>
			<td>PEO5-PPO70-PEO5</td>
		</tr>
		<tr>
			<td>Pluronic P123</td>
			<td>BASF</td>
			<td>&nbsp;</td>
			<td>PEO20-PPO70-PEO20</td>
		</tr>
	</tbody>
</table>
Table 1. Physico-chemical properties and designed concentrations (Molecular weight and Iso-electric point) of the selected peptide and protein standards.

low molecular weight protein enrichment on mesoporous silica thin films for biomarker discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

Watch this Scientific Journal Video about Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery at JoVE.com

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

low-molecular-weight-protein-enrichment-on-mesoporous-silica-thin

Research

JoVE Journal

Bioengineering

13.3K Views.  The Methodist Hospital Research Institute. We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Video: Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

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 ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...