The authors gratefully acknowledge Prof. Andrea Armani at the University of Southern California for support during the time this protocol was developed. Funding for the initial development of this work was provided by the National Science Foundation [085281 and 1028440] and the National Institute of Health through NIH Director's New Innovator Award Program [1DP2OD007391-01]. Additional information is available at <a href="http://web.missouri.edu/~hunthk/" target="_blank">http://web.missouri.edu/~hunthk/</a>.

1. Background
The biotin is attached to the surface of these devices through a simple, three-step process (Figure 1). First, we clean the surface and populate it with hydroxyl groups by exposing the devices to either oxygen plasma or piranha solution. Second, we use vapor deposition to attach the silane coupling agent terminated with a primary amine to the hydroxyl groups through hydrolysis and condensation reaction. Third, we attach biotin to the surface via N-hydroxysuccinimid...

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As described in the protocols, we created a housing platform by which to transport the silica microspheres by their stems throughout the functionalization process. This housing platform was created as a solution to the surface contamination and damage that resulted from the microsphere coming into contact with the walls of the various containers used throughout the functionalization process. We realized the main difficulty arose from constantly attaching and detaching individual microspheres to different containers durin...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>67-56-1</td>
			<td>ACS grade</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>Fisher</td>
			<td>8014-95-7</td>
			<td>Fuming</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Fisher</td>
			<td>7722-84-1</td>
			<td>30 wt %</td>
		</tr>
		<tr>
			<td>Aminopropyltrimethoxysilane</td>
			<td>Fisher</td>
			<td>13822-56-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NHS-biotin EZ linker</td>
			<td>Pierce</td>
			<td>20217</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Fisher</td>
			<td>67-68-5</td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Fluorescein Isothiocyanate</td>
			<td>Pierce</td>
			<td>46425</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher</td>
			<td>7647-14-5</td>
			<td>Powder concentrate</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate Buffer</td>
			<td>Fisher</td>
			<td>NC0099321</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Texas Red - Avidin Conjugate</td>
			<td>Pierce</td>
			<td>A820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optical Fiber</td>
			<td>Newport</td>
			<td>F-SC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Stripper</td>
			<td>Fiber Instrument Sales</td>
			<td>NN-175</td>
			<td>No-Nik 175 um stripper</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Fisher</td>
			<td>06666A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bare Fiber Cleaver</td>
			<td>Ilsintech</td>
			<td>Cl-03A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polypropylene Vials</td>
			<td>Fisher</td>
			<td>03-341-75A</td>
			<td>60 mL, hinged cap</td>
		</tr>
		<tr>
			<td>Incubating Rocker</td>
			<td>VWR</td>
			<td>12620-910</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>Fisher</td>
			<td>08-594-15B</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

attaching biological probes to silica optical biosensors using silane coupling agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Watch this Scientific Journal Video about Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents at JoVE.com

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

attaching-biological-probes-to-silica-optical-biosensors-using-silane

Research

JoVE Journal

Bioengineering

12.8K visualizações.  University of Missouri. Biossensores interface com ambientes complexos e biológicas e realizar a detecção de alvo, combinando sensores altamente sensíveis com sondas altamente específicos ligados ao sensor através de modificação da superfície. Aqui, nós demonstrar a funcionalização da superfície de sensores ópticos de sílica com biotina, utilizando agentes de acoplamento de silano para colmatar o sensor eo ambiente biológico.