Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/553—Metal or metal coated

Definitions

• the present invention relates generally to a biosensor, and in particular to a biocompatible nanowire scaffold usable for detecting one or more proteins of a biological analyte and supporting the growth of cells.
• Redox proteins such as heme proteins have inherent electrochemical activity and are therefore capable of exchanging electrons directly with a working electrode to produce an electrochemical signal. This allows direct electrochemical detection and quantification without a requirement for a mediator means, such as via an enzyme mediated reaction.
• biosensing devices for quantifying redox proteins which are not only accurate, but are also conveniently portable, disposable and able to analyze a biological sample which has undergone minimal pre-treatment would be of great interest for advancing the existing technologies in multiplex real-time, label-free detection of proteins.
• the present invention relates to a biocompatible scaffold.
• the biocompatible scaffold includes a substrate, and a conductive layer of Ti ⁇ 2 -containing nanowires or nanofibers formed on the substrate, where the conductive layer of TiCVcontaining nanowires or nanofibers is formed with a pore structure, and when the biocompatible scaffold is in contact with a biological analyte, one or more proteins of the biological analyte are immobilized on a surface of the conductive layer of TiCVcontaining nanowires or nanofibers so as to generate a measurable faradic current signal.
• the TiCh-containing nanofibers or nanowires are substantially in the TiCh-B phase or titanate phase, and have a typical diameter in the range of about 20-150 nm.
• the pore structure has a thickness in the range of about 1- 50 ⁇ m.
• the nanowire or nanofiber structure may have a layered-titanate (Na 2 Tis ⁇ 7) structure with counter-cations (Na + ) in the interlayer space.
• the substrate is formed of indium-tin-oxide (ITO) or a metal.
• the proteins comprise proteins with a redox-active center.
• the redox-active heme proteins comprise a cytochrome c.
• the substrate can be conductive or semi-conductive.
• the substrate is formed of indium-tin-oxide (ITO).
• ITO indium-tin-oxide
• the present invention relates to a biocompatible scaffold.
• the biocompatible scaffold has a substrate, and a layer of nanowires or nanofibers formed on the substrate.
• the layer of nanowires or nanofibers is formed with a pore structure.
• the nanowires or nanofibers comprise oxide-containing nanowires or nanofibers.
• the nanowires or nanofibers include TiCVcontaining nanowires or nanofibers.
• the present invention relates to a biosensor for detecting one or more proteins in a biological analyte.
• the biosensor includes a substrate having a surface, a layer formed of nanowires or nanofibers on the surface of the substrate, where the layer of nanowires or nanofibers is formed with a pore structure, and a detector in communication with the substrate, where when the layer formed of nanowires or nanofibers is in contact with one or more proteins in the biological analyte, a measurable signal is generated and measured by the detector.
• the biosensor further includes means for applying a potential to the biological analyte at a scan rate that is in the range of about 0.005-0.500 V/s.
• the detector comprises a cyclic voltammetry (CV).
• CV cyclic voltammetry
• the one or more proteins in the biological analyte contain redox-active heme proteins.
• the redox-active heme proteins include cytochrome c.
• the biological analyte contains a buffer solution having a pH value in the range of about 6.2-9.0, such that the cytochrome c carries a net positive charge, and the nanowires or nanofibers carry a net negative charge, respectively.
• the nanowires or nanofibers are oxide-containing nanowires or nanofibers.
• the nanowires or nanofibers are TiCVcontaining nanowires or nanofibers, where the Ti ⁇ 2 -containing nanowires or nanofibers are substantially in the Ti(VB phase or titanate phase.
• the nanowires or nanotubes have a typical diameter in the range of about 20-150 nm.
• the substrate is formed of indium-tin-oxide (ITO).
• ITO indium-tin-oxide
• the substrate can be conductive, or semi-conductive.
• the present invention relates to a method for detecting one or more proteins in a biological analyte.
• the method includes the steps of providing a biosensor having at least one electrode and a scaffold formed of nanowires or nano fibers; introducing the biological analyte into the scaffold; and detecting electron transfers between the biological analyte and the surface of the scaffold so as to detect one or more proteins in the biological analyte.
• the detecting step in one embodiment, is performed with a CV.
• the method further includes the step of applying a potential to the biological analyte at a scan rate that is in the range of about 0.005-0.500 V/s.
• the biological analyte contains redox-active heme proteins, where the redox-active heme proteins comprise cytochrome c.
• the biological analyte further contains a buffer solution having a pH value in the range of about 6.2-9.0, such that the cytochrome c carries a net positive charge, and the nanowires or nanofibers carry a net negative charge, respectively.
• Fig.1 shows schematically a biosensor according to one embodiment of the present invention
• Fig. 3 shows the characteristic of the titanate NW scaffold shown in Fig. 2,
• Fig. 4 shows the characteristic of the titanate NW scaffold shown in Fig. 2, (a) an SEM image of the PC- 12 cell grown on the titanate NW scaffold after cultured on the titanate NW scaffold for about 72 hours, and (b) CV curves of the cytochrome c immobilized onto the titanate NW scaffold at different potential scan rates and pH values;
• Fig. 5 shows the characteristic of an NW scaffold according one embodiment of the present invention.
• Fig. 6 shows the characteristic of an NW scaffold according one embodiment of the present invention.
• isoelectric point or its acronym “pi” refers to the pH at which a molecule or surface carries no net electrical charge.
• a molecule or surface must be amphoteric, meaning it must have both acidic and basic functional groups. Proteins and amino acids are common molecules that meet this requirement.
• Proteins can be separated according to their isoelectric point in a process known as isoelectric focusing. At a pH below the pi, proteins carry a net positive charge. Above the pi they carry a net negative charge. This has implications for running electrophoretic gels.
• the pH of an electrophoretic gel is determined by the buffer used for that gel. If the pH of the buffer is above the pi of the protein being run, the protein will migrate to the positive pole (negative charge is attracted to a positive pole). If the pH of the buffer is below the pi of the protein being run, the protein will migrate to the negative pole of the gel (positive charge is attracted to the negative pole). If the protein is run with a buffer pH that is equal to the pi, it will not migrate at all.
• cyclic voltammetry or its acronym “CV” refers is a type of potentiodynamic electrochemical measurement. To obtain a cyclic voltammogram, the voltage is varied in a solution and the change in current is measured with respect to the change in voltage. It is a specific type of voltammetry used for studying the redox properties of chemicals and interfacial structures.
• a potential is applied to the system, and the faradic current response is measured (a faradic current is the current due to a redox reaction).
• the current response over a range of potentials (a potential window) is measured, starting at an initial value and varying the potential in a linear manner up to a pre-defined limiting value.
• a switching potential the direction of the potential scan is reversed, and the same potential window is scanned in the opposite direction. This means that, for example, species formed by oxidation on the first (forward) scan can be reduced on the second
• Biocompatible scaffolds are found important in regenerations of tissue [1], such as bone [3, 4], under the guidance of proteins such as growth hormones.
• tissue [1] such as bone [3, 4]
• proteins such as growth hormones.
• an electrochemical protein sensor having a scaffold that enables fast electron transfer between the proteins and sensor electrodes.
• scaffolds of peptides and/or polymers usually have a poor electrical conductivity.
• NWs Solid-state nanowires
• TiCVbased NWs are inexpensive to fabricate, highly biocompatible, chemically and photochemically stable, with negligible protein denaturation, thus being widely used in various applications [6-8].
• heme proteins have been immobilized on TiO nanoparticles and nanotubes (NTs) [9-14].
• the present invention discloses a biocompatible scaffold of titanate-NWs, which entangle each other on the surface of an indium-tin-oxide (ITO) substrate (electrode).
• ITO indium-tin-oxide
• the titanate-NW scaffold is utilized as a biosensor, or at least a part of it, for monitoring the electrochemical redox potentials of the cytochrome c (cyt c) that is pre-immobilized on the titanate-NW's surface.
• titanate-NW scaffold can also be utilized to support the growth of pheochromocytoma cells (PC-12).
• this invention in one aspect, relates to a biocompatible scaffold usable for sensing proteins in a biological analyte and for supporting and directing the growth of stem cells.
• the biocompatible scaffold includes a substrate, and a conductive layer of Ti ⁇ 2 -containing nanowires or nanofibers formed on the substrate.
• the substrate can be conductive or semi-conductive.
• the substrate is formed of indium-tin-oxide (ITO) in one embodiment.
• ITO indium-tin-oxide
• the conductive layer of Ti ⁇ 2 -containing nanowires or nanofibers is formed with a pore structure having a thickness in the range of about 1- 50 ⁇ m.
• the pore structure may have a layered-titanate (TSfeTisO?) structure with counter-cations (Na + ) in the interlayer space.
• TfeTisO layered-titanate
• the Ti ⁇ 2 -containing NWs are substantially in the Ti ⁇ 2 -B phase or titanate phase, and have a typical diameter in the range of about 20- 150 nm.
• NWs containing the interlayer cations can also be utilized to practice the present invention.
• the biocompatible scaffold When the biocompatible scaffold is in contact with a biological analyte, one or more proteins of the biological analyte are immobilized on a surface of the conductive layer of Ti ⁇ 2 -containing nanowires or nanofibers, whereby electron transfers between the biological analyte and the surface of the conductive layer of Ti ⁇ 2-containing nanowires or nanofibers occur. The electron transfers generate a measurable faradic current signal.
• the biocompatible scaffold can be utilized to detect redox-active heme proteins of a biological analyte. Cytochrome c (cyt c) is a redox-active heme protein, has 104 amino acids, and sits in the intermembrane space of mitochondria as an electron carrier in the electron transport chain.
• cytochrome c carries a net positive charge, while the nanowires or nanofibers carry a net negative charge.
• a buffer solution having a pH value in the range of about 6.2-9.0 the cytochrome c carries a net positive charge, while the nanowires or nanofibers carry a net negative charge.
• the cytochrome c is immobilized on the surface of the TiCVcontaining NW scaffold, one or more electrons transfer between the cytochrome c and the NW scaffold is to cause a measurable signal in the form of a faradic current, which is detected by a cyclic voltammetry (CV).
• CV cyclic voltammetry
• Other types of proteins can also be utilized to practice the present invention. Referring to Fig. 1, a biosensor 100 utilizing the above NW scaffold 120 for detecting one or more proteins in a biological analyte is shown according to one embodiment of the present invention.
• the biosensor 100 has a substrate 110 having a surface 112, a scaffold 120 formed of NWs on the surface 112 of the substrate 110, and a detector (not shown) in communication with the substrate 110.
• the NW scaffold 120 is in contact with the biological analyte containing redox-active heme proteins 130 and a buffer solution.
• the buffer solution has a pH value in the range of about 6.2-9.0.
• the redox-active heme proteins such as cyt c 130 are immobilized on the surfaces of the NWs 120.
• the binding of cyt c on the NW scaffolds is attributed mainly to the electrostatic interaction between the negatively charged NW surface and the positively charged cyt c surface.
• the NW surface is negatively charged due to its isoelectric point of 6.2 [21], while the cyt c surface is positively charged due to its isoeletcric point of 10.0-10.5.
• the negative NWs surface shows a high affinity to the positive cyt c.
• the biosensor 100 also includes means (not shown) for applying a potential to the biological analyte at a scan rate that is in the range of about 0.005- 0.500 V/s.
• the detector in communication with the substrate 110 is used to detect electron transfers between the biological analyte and the surface 112 of the substrate 110.
• the detector includes a CV.
• the electron transfers are measured in terms of faradic current, i p .
• the presence and/or quantity of the proteins in the biological analyte can be determined by the faradic current i p
• the NW scaffold can also be utilized to support the growth of cells, such as pheochromocytoma cells (PC- 12).
• the NW scaffold is at least partially coated with a plurality of biomolecules, including growth hormone.
• Another aspect of the present invention relates to a method for detecting the one or more proteins of a biological analyte.
• the method includes the following steps: at first, a biosensor is provided, which has an electrode and a scaffold formed of NWs on the electrode. The biological analyte is then introduced into the scaffold. Electron transfers between the biological analyte and the surface of the substrate are detected by a CV, which is used determine the presence and/or quantity of the proteins in the biological analyte.
• titanate-NWs are entangled on the surface of an ITO substrate into a scaffold, which is used to monitor the electrochemical redox potentials of the cytochrome c that is pre-immobilized onto the titanate-NWs' surface.
• the titanate-NWs are synthesized at temperature about 240 0 C for about 3 days.
• Figs. 2a and 2b show a scanning electron microscope (SEM) image and a transmission electron microscopy (TEM) image of the titanate NW scaffold, respectively.
• SEM scanning electron microscope
• TEM transmission electron microscopy
• a powder x-ray diffraction (XRD) pattern of the titanate NW scaffold is shown in Fig. 2c. The XRD pattern clearly demonstrates that the crystal structure of the NWs belongs to the titanate phase (i.e.
• the edge-shared TiO 6 -octahedron is the basic unit to form the negatively charged, layered titanate structure [15], with the sodium counter-cations (Na + ) sitting in the interlayer space, thus resulting in variable interlayer distances depending on the size and the hydration degree of the cations. Accordingly, the titanate NW scaffold has a pore structure, as shown in Figs. 2a and 2b. After washing with the deionized distilled water (DDW to pH near 7), the sodium counter-cations are replaced with the protons.
• the resistance of the air-dried NW-scaffold is above 10 6 Ohm, which may not be suitable for the electrochemical sensing.
• the hydroxyl (- OH) groups [4] on the NW surface at a buffer solution of pH 6.2-9 could act as "wet- electrons", at the water-NW interface [16], to form a low-energy path for a fast ET on the NW surface, which ensures the signal transduction(s) across the entire biocompatible scaffold in biosensing.
• the surface of titanate NWs may be negatively charged and formed Ti-OH in the buffer solution [4].
• the interface between water and metal-oxide can produce wet-electrons [16], thus, the conductivity of titanate-NWs is improved.
• Cytochrome c is a redox-active heme protein, has 104 amino acids, and sits in the intermembrane space of mitochondria as an electron carrier in the electron transport chain.
• FT-IR Fourier transform infrared spectroscopy
• curve 210 is the FT-IR spectrum for cytochrome c-titanate- NWs mixed with KBr
• curve 220 is the FT-IR spectrum for cytochrome c in the pH 6.8 buffer solution.
• the FT-IR spectra show a peak at 1648 cm "1 for amide I [20] of immobilized cyt c on the NWs, which is same as that obtained from native cyt c in buffer of pH 6.8, showing the retained secondary structure for the cyt c immobilized on the titanate-NW scaffold.
• the binding of cyt c on the titanate-NW scaffolds are attributed mainly to the electrostatic interaction between the negatively charged NW surface and the positively charged cyt c surface.
• the NW surface is negatively charged due to its isoelectric point of 6.2 [21], while the cyt c surface is positively charged due to its isoeletcric point of 10-10.5.
• the negative NWs surface shows a high affinity to the positive cyt c.
• the NW scaffold offers a desirable environment for the cyt c to undergo facile electron-transfer reactions.
• the surface electrochemical properties of the NWs immobilized with cytochrome c are studied by means of a CV at various concentrations, which indicates a detection limit of 45 picomoles (or 10 "12 moles) of the cyt c.
• the scan rate is about 0.2V/s.
• a pair of reversible and well defined redox peaks from the cyt c-NW electrode with the formal peak potential (E 0 ) of 0.03 V have been recorded.
• the E 0 value (0.03 V), calculated from an average of anodic peak's and cathodic peak's potential values, is close to that reported by Stellwagen [23]. Based on its structure and morphology, the NWs behave like a conducting "nanocable" [16] to facilitate the electron transfers.
• Measurement of the faradic current as a function of the scan rate can be used to diagnose whether a redox reaction on the electrode surface is controlled by diffusion.
• the cathodic and the anodic current peaks are both linearly proportional to the scan rate from 0.01 V/s to 0.2 V/s, implying that such an electrode has the typical characteristic of the thin-layer electrochemistry [24].
• the ratio of i pc to i pa is about 2.0 and the separations between the redox peaks was about 58 mV (data not shown), indicating that the electrochemical process on the cyt c-NW electrode surface may be quasi-reversible [24].
• the faradic current is increased linearly as the concentration of the cyt c in the scaffolds was increased, as shown in the Fig. 3b, where signal 351a is i pa for 45 picomoles, signal 352a is i pa for 122 picomoles, signal 353a is i pa for 243 picomoles, signal 354a is i pa for 347 picomoles and signal 355a is i pa for 450 picomoles, respectively, and the scan rate is about 0.2V/s.
• the reversible, well-defined CV signals are observed in 15 ⁇ L solutions between 3.0 ⁇ M and 30.0 ⁇ M, or 45 to 450 picomoles.
• the NW scaffold can also be utilized for supporting and directing the growth of pheochromocytoma cells (PC-12), thereby facilitating cellular activities.
• Figs. 3d and 4a are an SEM image of PC-12 cells cultured on the NW scaffold for about 72 hours, showing that PC-12 cells attached well and formed cell colonies on the NW scaffold. The cell appeared round in shape and maintained their morphologies, indicating a good compatibility between the cell and the NW scaffold. PC-12 cells attachment on the titanate-NW scaffold further suggests the adsorbed proteins do not lose any of their activities.
• Fig. 4b shows CV signals of protein cytochrome c immobilized onto a thin- film of the NW scaffold at different potential scan rates and pH values.
• a reproducible detection limit of 45 picomoles of the cytochrome c has been achieved at the range of pH 6.8-9.0, revealing a greatly enhanced direct electron transfer for the cytochrome c on the titanate NW-modified ITO electrode.
• the formal potential of the cytochrome c on the NW is independent to the pH change. The results would shed new light on fabricating the NW-based affinity-specific bionanosensors via a functionalization on the ceramic nanowire surface could result in a multiplexed sensing platform for simple, economic, quick, sensitive, and selective biosensing.
• Figs. 5 and 6 are CV signals of a NW scaffold according to another embodiment of the present invention.
• the present invention discloses a biocompatible scaffold of the titanate NWs, and a titanate NW scaffold based biosensor.
• the titanate NW scaffold enhances the electron transfers between the ITO electrode and the cyt c.
• the titanate NW scaffold based biosensor can find applications in direct detection of immobilized redox-active proteins and monitoring biochemical processes during the growth of stem cells in real time within a confined environment of the titanate NW scaffold based scaffold.

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