The authors would like to acknowledge Ph.D. grant of LANEF in Grenoble for support of Laurie-Amandine Gar&#231;on. This work was financially supported by the French National Research Agency (ANR-grant 06-NANO-045).

1. Preparation of Various Solutions and Protein Samples
<ol>
	<li>Prepare 100 ml of phosphate buffer solution (PBS-G) containing 50 mM NaH2PO4, 50 mM NaCl, and 10% glycerol at pH 6.8.</li>
	<li>Prepare 250 ml of HEPES buffer solution containing 10 mM HEPES, 150 mM NaCl, 0.005% Tween 20 at pH 7.4.</li>
	<li>Prepare stock solution of building block 1 (BB1) lactose and building block 2 (BB2) sulfated lactose (Figure 1)&#160;at 0.2 mM in PBS-G.</li>
	<li>Prepare 1 ml of protein solutions in HEPES: Arachis hypogaea lectin (AHL) at 500 nM, myoglobin at 1 &#181;M, and lysozyme at 500 nM.</li>
	<li>Prepare 20 ml of 1% SDS in ultrapure water.</li>
</ol>
2. Preparation of the CoCRR Array
<ol>
	<li>Clean the gold surface of the prism 48 hr prior to use with a plasma cleaner for 3 min under these conditions: 75% Oxygen, 25% Argon, 0.6 mbar, power 40 W.</li>
	<li>Prepare eleven pure and mixed solutions of BB1 and BB2 with [BB1]/([BB1]+[BB2]) ratios between 0 and 100% by increments of 10% at a total BB concentration of 0.1 mM in PBS-G.</li>
	<li>Deposit 8 nl droplets of these pure and mixed solutions on the prism surface using a non-contact spotter in quadruplicate for each ratio with 44 spots in total (Figure 2). 
	NOTE: 10% glycerol added in PBS-G is very important to reduce solvent evaporation and the change of BB concentration after deposition.</li>
	<li>Place the prism inside a Petri dish containing 1 ml of ultrapure water and leave it O/N at RT for self-assembly of BB1 and BB2 on the gold surface. Herein, it is assumed that the average surface composition in the mixed SAMs reflects the composition of the deposition mixed solution (Figure 3)11.</li>
	<li>Wash thoroughly the prism with ultrapure water and then dry it under a flow of argon.</li>
</ol>
3. Protein Sensing by SPRi
<ol>
	<li>Set the incubator in which the SPRi apparatus is placed at 25 &#176;C to avoid refractive index changes induced by temperature variation during protein sensing.</li>
	<li>Insert a non-functionalized rinsing prism in the 10 &#181;l Polyether ether ketone (PEEK) flow cell connected to a computer controlled syringe pump, a degasser and a 6-port medium pressure injection valve (Figure 4). Fill in the flow system with freshly filtered and degassed running buffer HEPES.</li>
	<li>Remove the rinsing prism and insert the prism containing the CoCRR array in the flow cell. Run HEPES at a 100 &#181;l/min flow rate. With the help of the CCD camera remove any air bubbles present on the prism surface by passing running buffer quickly.</li>
	<li>Define study area for each spot by drawing a circle with the same diameter for the area of interest based on a well-contrasted image of the array and with the help of integrated software in SPRi system.</li>
	<li>Trace plasmon curves, which represent reflectivity curves in function of the incident angle, for all the spots.</li>
	<li>Choose the working angle (position at which kinetic curves will be recorded) at the highest slope of the reflectivity curves. Rotate the scanning mirror to fix the selected working angle for kinetic measurements.</li>
	<li>Continue to run HEPES through the flow system until reflectivity signal for all the spots is stable and constant.</li>
	<li>Inject 1 ml AHL solution with a syringe into the sample loop (500 &#181;l) with the injection valve on position &#8220;load&#8221;. Then put the injection valve to position &#8220;injection&#8221;. The flow rate used for all the protein injection was 100 &#181;l/min.</li>
	<li>Start kinetic measurements by monitoring reflectivity variations against time simultaneously on all the spots.</li>
	<li>At the end of protein injection, rinse the array with running buffer for 8 min. Finally, inject 1 ml 1% SDS to regenerate it.</li>
	<li>Repeat the same procedure for the other proteins.</li>
</ol>
4. Data Processing and Analysis
<ol>
	<li>Following entry of protein solution into the flow cell, molecular binding occurs and induces a shift of the plasmon curves and a variation of reflectivity. The image acquisition software converts the measured light intensity values to gray scale levels, giving SPR images Figure 5A, and generates the variation of reflectivity versus time, giving sensorgrams (Figure 5B).</li>
	<li>Use a math computing software to plot the reflectivity (R%) at the end of each protein injection versus the [BB1]/([BB1]+[BB2]) ratio to generate 2D continuous evolution profile (CEP) for each sample (Figure 5C).</li>
	<li>Add the [BB1]/([BB1]+[BB2]) ratio in sensorgrams (Figure 5B)&#160;to generate 3D continuous evolution landscape (CEL) for each protein (Figure 5D).</li>
</ol>

<ol>
	<li>Margulies, D., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+protein+recognition+as+an+alternative+approach+to+antibody-mimetics.">Combinatorial protein recognition as an alternative approach to antibody-mimetics.</a> Current Opinion in Chemical Biology. 14, 705-712 (2010).</li><li>Umali, A. P., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+approach+to+differential+sensing+using+synthetic+molecular+receptors.">A general approach to differential sensing using synthetic molecular receptors.</a> Current Opinion in Chemical Biology. 14, 685-692 (2010).</li><li>Baldini, L., Wilson, A. J., Hong, J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern-based+detection+of+different+proteins+using+an+array+of+fluorescent+protein+surface+receptors.">Pattern-based detection of different proteins using an array of fluorescent protein surface receptors.</a> J. Am. Chem. Soc. 126, 5656-5657 (2004).</li><li>Zhou, H. C., Baldini, L., Hong, J., Wilson, A. J., Hamilton, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+of+proteins+based+on+an+array+of+functionalized+porphyrins.">Pattern recognition of proteins based on an array of functionalized porphyrins.</a> J. Am. Chem. Soc. 128, 2421-2425 (2006).</li><li>Wright, A. T., Griffin, M. J., Zhong, Z., McCleskey, S. C., Anslyn, E. V., McDevitt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptors+create+patterns+that+distinguish+various+proteins.">Differential receptors create patterns that distinguish various proteins.</a> Angew. Chem. Int. Ed. 44, 6375-6378 (2005).</li><li>Wright, A. T., Anslyn, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+receptor+arrays+and+assays+for+solution-based+molecular+recognition.">Differential receptor arrays and assays for solution-based molecular recognition.</a> Chem. Soc. Rev. 35, 14-28 (2006).</li><li>You, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+proteins+using+nanoparticle&#8211;fluorescent+polymer+&#8216;chemical+nose&#8217;+sensors.">Detection and identification of proteins using nanoparticle&#8211;fluorescent polymer &#8216;chemical nose&#8217; sensors.</a> Nature Nanotechnology. 2, 318-323 (2007).</li><li>De, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+proteins+in+human+serum+using+conjugates+of+nanoparticles+and+green+fluorescent+protein.">Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein.</a> Nature Chemistry. 1, 461-465 (2009).</li><li>Hou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+evolution+profiles+for+electronic-tongue-based+analysis.">Continuous evolution profiles for electronic-tongue-based analysis.</a> Angew. Chem. Int. Ed. 51, 10394-10398 (2012).</li><li>Campbell, C. T., Kim, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPR+microscopy+and+its+applications+to+high-throughput+analyses+of+biomolecular+binding+events+and+their+kinetics.">SPR microscopy and its applications to high-throughput analyses of biomolecular binding events and their kinetics.</a> Biomaterials. 28, 2380-2392 (2007).</li><li>Stranick, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanometer-scale+phase+separation+in+mixed+composition+self-assembled+monolayers.">Nanometer-scale phase separation in mixed composition self-assembled monolayers.</a> Nanotechnology. 7, 438-442 (1996).</li></ol>

The authors have no conflicts of interest to declare.

The most critical steps for construction of this eT are dedicated to ensure good reproducibility of the system. For example, cleaning the gold surface of the prism with a standardized procedure before use, adding 10% of glycerol in the eleven pure and mixed solutions of BB1 and BB2 to eliminate solvent evaporation during BBs self-assembling on the gold surface of the prism, depositing multiple replicates for each [BB1]/([BB1]+[BB2]) ratio,&#160;etc. As for protein sensing by SPRi, the choice of the working angle is critical. Usually, it is fixed at the highest slope of the reflectivity curves. In addition, it is very important to use standardized experimental conditions for each protein sensing, such as working temperature, flow rate,&#160;etc. After each protein injection, the CoCRR array must be regenerated for reuse with an appropriate solution that allows complete regeneration of the system without any damage to receptors.
To demonstrate that the developed eT is not only effective for the study of sugar binding proteins as previously reported9, but also for common proteins, one lectin AHL together with two non-sugar-binding proteins myoglobin and lysozyme were used. They were chosen to have a variety of charges in HEPES (pH 7.4): AHL (isoelectric point (pI) 6.0), myoglobin (pI 7.2) and lysozyme (pI 11). Notably, as shown in Figure 6, each protein possesses a unique response pattern. AHL is slightly negatively charged in HEPES. Its CEP shows that it has stronger interaction with the negatively charged BB2-rich CoCRRs. Most likely, it is due to the interaction between the positively charged domain of the protein and BB2-rich CoCRRs. For myoglobin, which is globally neutral in HEPES, there is not much difference between signals obtained with all CoCRRs. When comparing myoglobin in CEP to the CEPs of AHL and lysozyme, even at a higher concentration the signal is much lower. As for lysozyme, which is positively charged in HEPES, the maximal signal was obtained with the CoCRR containing pure BB2.
Thanks to the advantages of SPRi capable of monitoring the real-time adsorption and desorption kinetics, for each protein a 3D continuous evolution landscape was generated. This kind of time-dependent recognition pattern brings supplementary differentiation parameters for protein analysis. Particularly, it could be extremely useful for analyzing two analytes presenting the similar affinity for a set of CoCRRs but differing in their kinetics of interaction. As shown in Figure 7, the CELs are easily distinguishable. These results show that the interaction of the three proteins with the CoCRRs is dependent on their surface characteristics such as the distribution of hydrophobic, neutral and charged amino-acid residues. Both CEP and CEL can be used as &#8220;fingerprints&#8221; for the protein discrimination and prospective identification. Thus, the eT is effective for analysis of common proteins.
The protocol described herein uses a combinatorial approach to construct the eT for protein analysis. Unlike other approaches that use a number of molecules which are structurally complicated and laborious to synthesize, only two small molecules were used to prepare the cross-reactive receptor array capable of differentiating proteins with good resolution in present study. Additionally, according to the previous investigation, the eT is stable, reproducible and reusable9.&#160;Furthermore, SPRi allows monitoring binding events in real-time and generating novel continuous recognition patterns with unprecedented reliability. In the near future, additional BBs with complementary physicochemical properties will be introduced to increase the diversity of the CoCRR in the arrays for analysis of complex mixtures in liquid or in gas.

Precise and rapid protein sensing methods are very important in medical diagnostics and proteomics. Classical protein-detecting arrays, such as biochips, are based on the &#8220;lock-and-key&#8221; recognition principle and require specific receptors such as aptamers, antibodies, or mimetics.
In recent years, differential sensing inspired by the human olfaction and gustation has emerged as an alternative1. This electronic nose/tongue (eN/eT) approach is based on differential binding of analytes to an array of cross-reactive receptors (CRRs), that do not need to be highly specific or selective for the target molecules thus allow to surmount the laborious process of developing highly selective receptors. It is the combined response of all the receptors that creates a distinct pattern for each sample, like a fingerprint, allowing its identification.
The two key challenges for the development of electronic nose/tongue for effective protein sensing are the production of sensing elements that have the ability to distinguish among structurally similar analytes and the appropriate transduction system for the binding event. Up to now, studies have reported various approaches to array development2. For example in one study an array-based identification of proteins was developed using CRRs prepared from tetra-carboxyphenylporphyrin derivatives by coupling the carboxyl groups to various amino acids or dipeptides to provide differential receptors possessing a hydrophobic core for affinity for proteins and distinct charged peripheries for imparting differential binding. Using this system, different proteins and protein mixtures were identified by measuring fluorescence quenching of the receptors upon interaction with the analytes3,4. In another study, a library of 29 CRRs containing tripeptide and boronic acid moieties synthesized in a combinatorial way on a hexasubstituted benzene scaffold was developed for sensing proteins with an indicator-uptake colorimetric detection5,6. With such a design, each receptor showed differential binding capacity with proteins based on the variance in the peptide arms, and the boronic acids assisted in differentiation of proteins from glycoproteins. More recently, an array composed of different cationic functionalized gold nanoparticles conjugated with an anionic fluorescent polymer poly(p-phenyleneethynylene) (PPE) has been created to detect and identify proteins7. The competitive binding between protein analytes and quenched PPE/gold nanoparticle complexes regenerated fluorescence, producing distinct recognition patterns for proteins. In this study, the functionalized nanoparticle-protein interactions were tuned by varying physicochemical properties of nanoparticle end groups. Furthermore, it was shown that this approach is effective for protein analysis in complex and protein-rich medium such as human serum at physiologically relevant concentrations, thus showing the potential of eT in profiling real samples for diagnosing disease states8.
Though very promising, these systems have some inherent limitations. They require designing and synthesizing from 5 to 29 CRRs with quite complicated structures. In addition, unlike the olfactory system that is reset following each measurement, protein sensing requires preparing an array per sample. Finally, monitoring real-time binding events are extremely difficult.
In this context, a combinatorial approach was proposed by using a small number of simple and easily accessible molecules with different physicochemical properties (hydrophilic, hydrophobic, positively charged, negatively charged, neutral, etc.) as building blocks (BBs)9. By mixing BBs in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for binding protein was created. Notably, the self-assembled monolayers on this system allow easy tuning of a range of surface properties in a highly divergent fashion, enabling diverse combinatorial cross-reactive receptors (CoCRRs) to be rapidly and efficiently produced. Protein sensing was performed using an optical detection system, surface plasmon resonance imaging (SPRi). Briefly, a broad-beam monochromatic polarized light from a LED illuminates the whole CoCRR array area on the surface of the prism. A high resolution CCD video camera provides real-time difference images across all the spots of the CoCRR array. It captures all of the local changes at the surface of the CoCRR array providing detailed information on binding events and kinetic processes10. Meanwhile, with the help of imaging software, SPR images corresponding to spots are automatically converted to variations of reflectivity versus time, generating a series of kinetic binding curves called sensorgrams. Thus, SPRi allows a label-free, synchronous, parallel, and real-time observation of binding events. Additionally, the obtained CoCRR array is regenerable and reusable for protein analysis.
This protocol describes the construction of the electronic tongue by using only two small molecules as building blocks and illustrates its application for analysis of common proteins based on continuous recognition patterns obtained with SPRi.

<table border="0" cellpadding="0" cellspacing="0" width="1511">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:225px;">Catalog Number</td>
			<td style="width:799px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPRi apparatus&#160;</td>
			<td>Horiba Scientific-GenOptics</td>
			<td style="width:225px;"></td>
			<td>SPRi apparatus is placed in a temperature regulated incubator at 25&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Memmert</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe pump&#160;</td>
			<td>Cavro scientific instruments</td>
			<td style="width:225px;">Cavro XLP 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">Micro Elite Degasser&#160;</td>
			<td style="width:203px;">Alltech</td>
			<td style="width:225px;">AT590507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-port medium pressure injection valve</td>
			<td>Upchurch Scientific</td>
			<td style="width:225px;"></td>
			<td>The volume of injection loop used is 500 &#181;l.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Femto plasma cleaner (version 7)</td>
			<td>Diener Electronic</td>
			<td style="width:225px;"></td>
			<td>On-line degassing system with 2 channel.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spotter</td>
			<td style="width:203px;">Siliflow</td>
			<td style="width:225px;"></td>
			<td>&#160;It is a non-contact piezoelectric spotter.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SPRi-Biochip</td>
			<td>Horiba Scientific-GenOptics</td>
			<td align="right" style="width:225px;">36000067</td>
			<td>The prism is made of a high refractive index glass prism coated with a thin gold film (45 nm) and specially developed for imaging purposes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">erythrina cristagalli lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L5390</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">arachis hypogaea lectin&#160;</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L0881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">myoglobin</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">M1882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lysozyme</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#945;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">CXCL12&#947;&#160;</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Dr. Hugues Lortat-Jacob, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sulfated lactose</td>
			<td style="width:203px;"></td>
			<td style="width:225px;"></td>
			<td>Provided by Prof. David Bonnaff&#233;, for preparation details, see supporting information in reference 9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">glycerol</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">G5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">SDS</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">tween 20</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td align="right" style="width:225px;">274348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">HEPES</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium phosphate monobasic</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S0751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:285px;">sodium chloride</td>
			<td style="width:203px;">Sigma-Aldrich</td>
			<td style="width:225px;">S3014</td>
			<td></td>
		</tr>
	</tbody>
</table>

electronic tongue generating continuous recognition patterns for protein analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

To probe the ability of the electronic tongue for common protein analysis, three proteins were used: AHL, myoglobin and lysozyme. For each protein, a distinct 2D continuous evolution profile, CEP, was generated by the eT, as shown in Figure 6.
In addition, thanks to SPRi, which is able to monitor the real-time adsorption and desorption kinetics, for each protein a time dependent continuous recognition pattern, called 3D continuous evolution landscape (CEL), was generated. In Figure 7, characteristic CELs of these three proteins are shown.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig1highres.jpg" width="500" /> 
Figure 1. Chemical structures of the two building blocks used in this protocol to prepare the CoCRR array. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51901/51901fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig2highres.jpg" width="500" /> 
Figure 2. A photo of a prism after depositing the 44 droplets of the pure and mixed solutions of BB1 and BB2 using a non-contact piezoelectric spotter with each ratio in quadruplicate. The prism was placed next to an Eppendorf tube of 1.5 ml.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig3highres.jpg" width="500" /> 
Figure 3. Schematic illustration of the CoCRR array containing 11 differential receptors that were prepared with pure and mixed solutions of BB1 and BB2 at various ratios and allowing the mixtures to self-assemble on the gold surface of the prism.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig4highres.jpg" width="500" /> 
Figure 4. Schematic illustration of the SPRi detection system combined with a microfluidic system.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig5highres.jpg" width="500" /> 
Figure 5. Schematic illustration of data treatment for generation of two types of continuous recognition pattern with developed electronic tongue: 2D continuous evolution profile and 3D continuous evolution landscape.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig6highres.jpg" width="500" /> 
Figure 6. Continuous evolution profiles for three pure proteins: AHL (500 nM), myoglobin (1 &#181;M), and lysozyme (500 nM). They were generated by plotting the variation of reflectivity (R%) at the end of protein injection versus the [BB1]/([BB1]+[BB2]) ratio.
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51901/51901fig7highres.jpg" width="500" /> 
Figure 7. Landscape of taste: characteristic 3D CELs of AHL, myoglobin and lysozyme generated by the electronic tongue.

Watch this Scientific Journal Video about Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis at JoVE.com

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

electronic-tongue-generating-continuous-recognition-patterns-for

Nanyang Technological University

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JoVE Journal

Bioengineering

7.8K Views.  Institut Nanosciences et Cryogénie, CEA-Grenoble. A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination. 

Video: Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

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

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

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

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...