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"Electroadsorption and Charge Based Biomolecule Separation and Detection in Porous Sensors" in Patent Application Approval Process

July 8, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent application by the inventors Sailor, Michael J. (La Jolla, CA); Chen, Michelle Y. (Anaheim, CA), filed on May 2, 2012, was made available online on June 26, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also The Regents Of The University Of California).

This patent application is assigned to The Regents Of The University Of California.

The following quote was obtained by the news editors from the background information supplied by the inventors: "The fidelity of detection in a biosensor is limited by its ability to identify small quantities of analyte in the presence of substantial and often much larger quantities of interfering molecules. Separation, preconcentration, and detection of the analyte are key aspects of the analysis. The drive to decrease sample volumes and increase throughput has led to chip-based microanalysis systems that combine these components within a volume of a few cubic micrometers.

"Electric fields, applied via external electrodes or photogenerated in a semiconducting matrix, are often employed to enhance biomolecular separation in such systems. For example, electroadsorption typically concentrates a charged analyte on a solid metal electrode surface or on liquid mercury, and electrophoresis induces migration and separation of charged species.

"Early work demonstrated single-molecule transport in a nanopore constructed from a natural membrane protein. Following that work other nanoscale porous structures have been developed, such as .alpha.-haemolysin (Bayley, H. et al., 'Stochastic Sensors Inspired by Biology' Nature 413, 226-230 (2001)), artificial polymeric (Howorka, S. et al. 'Nanopore Analytics: Sensing of Single is Molecules' Chemical Society Reviews 38, 2360-2384 (2009)), inorganic (Howorka et al [supra]; Li, J. et al., 'Ion-beam Sculpting at Nanometre Length Scales' Nature 412, 166-169 (2001); Striemer, C. C. et al., 'Charge- and Size-Based Separation of Macromolecules Using Ultrathin Silicon Membranes' Nature 445, 749-753 (2007)), and composite (Wanunu, M. et al., 'Chemically-Modified Solid State Nanopores' Nano. Lett. 7 (2007)).

"Carbon nanotubes and other nanostructures have also been used for molecule transport. The constricted environment in a nanopore has a substantial influence on molecular transport that can be harnessed for biosensing. (Choi, Y., et al., 'Biosensing with Conically Shaped Nanopores and Nanotubes' Phys. Chem. Chem. Phys. 8, 4976-4988 (2006); Siwy, Z. et al., 'Protein Biosensors Based on Biofunctionalized Conical Gold Nanotubes' J. Am. Chem. Soc. 127, 5000-5001 (2005)) filtration (Streimer et al. [supra]; Han, J., et al., 'Molecular Sieving Using Nanofilters: Past, Present and Future' Lab Chip 8, 23-33 (2008).

"Diffusion or migration of biomolecules is often accomplished by applying an electric field. This technique has been used to enhance the rate and selectivity of ionic transport in various microfluidic devices (Li, Q. et al., 'Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS,' Anal. Chem. 81, 2242-2250 (2009)) and nanotube membranes (Yu, S. et al., 'Electrophoretic Protein Transport in Gold Nanotube Membranes,' Anal. Chem. 75, 1239-1244 (2003)).

"Such electrophoretic transport through membranes requires significant voltages to generate the necessary electric field strength. Typically greater than 1 KV applied voltage is needed to achieve field strength in the range of 100 V/cm to 500 V/cm. Because of the high voltages needed to produce electrophoretic transport the electrodes in these experiments are usually far removed from the separation matrix to avoid excessive heating or degradation of the analyte molecules. (Li, Q. et al [supra]). Charged molecules can be moved is with significantly smaller voltages (Gurtner, C., et al., 'Photoelectrophoretic Transport and Hybridization of DNA Oligonucleotides on Unpatterned Silicon Substrates,' J. Am. Chem. Soc. 122, 8589-8594 (2000)).

"Electroadsorption is a well-established means to concentrate analytes (including biologicals) on electrode surfaces that are then forwarded downstream to a separate detector (Wandlowski et al. [supra]; Ban, A., et al., 'Fundamentals of Electrosorption on Activated Carbon for Wastewater Treatment of Industrial Effluents,' J. Appl. Electrochem. 28, 227-236 (1998); Koresh, J. et al., 'Stereoselectivity in Ion Electroadsorption and in Double-Layer Charging of Molecular-Sieve Carbon Electrodes' J. Electroanal. Chem. 147, 223-234 (1983); Salitra, G., et al., 'Carbon Electrodes for Double-Layer Capacitors--I. Relations Between Ion and Pore Dimensions,' J. Electrochem. Soc. 147, 2486-2493 (2000). Electroadsorption involves the adsorption of ionized species onto an electrode surface upon application of small potentials (typically

"Membranes with abiotic micro- or nanometer sized pores have been used for sensing, filtration, controlled release and gating of biological molecules. They are typically considered to be more stable and predictable than biological pores. The properties of the membranes provide a range of synthetic alternatives to control analyte transport behavior, sensitivity, and specificity. Porous silicon (pSi) membranes offer a versatile platform for studies of protein transport and binding: the porous nanostructure can be controlled during synthesis to yield a range of pore sizes, and optical structures can be incorporated into the films to provide sensitive, label-free quantification of biomolecules.

"Porous silicon films have been previously demonstrated to separate biomolecules based upon size and negative native charge of the surface of oxidized porous silicon. See, e.g., Kosmulski, 'In Adsorption on Silica Surfaces,' Papirer, E., Ed.; Marcel Dekker: New York, 2000; Vol. 90, pp 363-364; Sailor et al., 'Sustained Release of a Monoclonal Antibody from Electrochemically Prepared Mesoporous Silicon Oxide,' Adv. Funct. Mater. 2010, 20, 4168-4174. Although both pore size and the charge on the pore walls should have an effect on transport and binding of proteins, the transport of charged proteins within such porous silicon films has not been studied. The time dependence of protein transport in mesoporous SiO.sub.2 films has been observed but not addressed or quantified. See, e.g., Sailor et al., 'Confinement of Thermoresponsive Hydrogels in Nanostructured Porous Silicon Dioxide Templates,' Adv. Funct. Mater. 2007, 17, 1153-1162; Sailor et al., 'Confinement of Thermoresponsive Hydrogels in Nanostructured Porous Silicon Dioxide Templates,' J. Am. Chem. Soc. 2005, 127, 11636-11645. In addition, explicit control of the gating mechanism is not believed to have previously been addressed."

In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors' summary information for this patent application: "The present invention provides electroadsorption and charged based biomolecule separation, concentration and detection with porous biosensors. In preferred embodiments, a potential is applied to a porous electrode to separate and concentrate molecules from solution. The biomolecular analytes are captured by the porous electrode itself, the same electrode that is used to generate the electric field for electroadsorption. In additional preferred embodiments, pH of the solution is adjusted to separate and concentrate biomolecules. Setting the pH equal to the protein isoelectric point was determined by the inventors to maximize concentration of biomolecules into the porous biosensor. The methods include simultaneously optically detecting charged molecules captured by the porous electrode. Methods of the invention are benign to biomolecules of interest, which are demonstrated to retain a high percentage of their activity after being released from the biosensor. Methods of the invention provide label-free detection. Advantageously, small voltages and ultrasmall volumes of solution are used in methods of the invention.

BRIEF DESCRIPTION OF DRAWINGS

"FIGS. 1A (top-plan) and 1B (cross-section) show experimental images of a porous Si sample prior to carbonization;

"FIG. 1C shows a light spectrum of a carbonized pSi sample immersed in pH 6.7 buffer;

"FIG. 1D shows the Fourier transformed spectrum of FIG. 1C;

"FIG. 2A illustrates voltage dependent adsorption of lysozyme, specifically the percentage change in optical thickness as a function of time for a carbonized pSi film as lysozyme is adsorbed under control of electrical bias values that are applied;

"FIG. 2B illustrates concentration factors representing the amount of lysozyme loaded into the carbonized pSi film relative to the bulk solution concentration;

"FIGS. 3A-3C show optical responses of carbonized pSi sensors upon application of bias in the presence of lysozyme as a function of time;

"FIG. 4 illustrates the percentage change in lysozyme concentration as function of applied voltage in response to discrete voltage steps from -0.5 to -2.75 V;

"FIG. 5A shows current transient after application of a -0.5 V step to the pSi film and FIG. 5B shows the natural logarithm of the current vs time trace;

"FIGS. 6A-6C show temporal responses of optical carbonized pSi sensors to lysozyme with applied bias as a function of ionic strength and applied bias voltage;

"FIG. 7 includes data representing lysozyme activity after interaction with carbonized pSi films with electroadsorption;

"FIGS. 8A-8D show the temporal optical response of a pSiO.sub.2 film upon introduction of bovine serum albumin (BSA);

"FIGS. 9A-9B show a comparison of infiltration of the proteins BSA, bovine hemoglobin (BHb) and equine myoglobin (EMb) into a pSiO.sub.2 film;

"FIGS. 10A and 10B compare the temporal response of BSA infiltration at protein isoelectric point pI=pH (4.7) and pI>pH (4.2) to theoretical curves derived from Fick's Second law;

"FIGS. 11A-11C model the extent of protein infiltration and the rate at which it is admitted to a mesoporous pSiO.sub.2 film at different pH values relative to protein isoelectric point (p1);

"FIG. 12 is the IEF (isoelectric focusing) gel electrophoresis data with isoelectric pH as indicated on the left for of BSA, BHb, and EMb;

"FIGS. 13A and 13B are DLS (dynamic light scattering) showing the hydrodynamic diameter of BSA, BHb, and EMb as a function of pH obtained on filtered protein solutions; and

"FIGS. 14A and 14B show the influence of solution ionic strength on extent of infiltration and zeta potential of BSA."

URL and more information on this patent application, see: Sailor, Michael J.; Chen, Michelle Y. Electroadsorption and Charge Based Biomolecule Separation and Detection in Porous Sensors. Filed May 2, 2012 and posted June 26, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=7313&p=147&f=G&l=50&d=PG01&S1=20140619.PD.&OS=PD/20140619&RS=PD/20140619

Keywords for this news article include: Lysozyme, Nanotube, Chemicals, Chemistry, Biosensing, Bioengineering, Porous Silicon, Silicon Dioxide, Bionanotechnology, Nanobiotechnology, Emerging Technologies, The Regents Of The University Of California.

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Source: Life Science Weekly


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