+

WO2008030395A1 - Appareil et procédé de détermination quantitative de molécules cibles - Google Patents

Appareil et procédé de détermination quantitative de molécules cibles Download PDF

Info

Publication number
WO2008030395A1
WO2008030395A1 PCT/US2007/019166 US2007019166W WO2008030395A1 WO 2008030395 A1 WO2008030395 A1 WO 2008030395A1 US 2007019166 W US2007019166 W US 2007019166W WO 2008030395 A1 WO2008030395 A1 WO 2008030395A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanowires
nanoscale wires
electronic device
molecules
target
Prior art date
Application number
PCT/US2007/019166
Other languages
English (en)
Inventor
Michael Amori
Yuri Bunimovich
James R. Heath
Young Shik Shin
Original Assignee
California Institute Of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by California Institute Of Technology filed Critical California Institute Of Technology
Publication of WO2008030395A1 publication Critical patent/WO2008030395A1/fr

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
    • G01N27/4146—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS involving nanosized elements, e.g. nanotubes, nanowires
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip

Definitions

  • the present disclosure relates to determination of molecules.
  • it refers to an apparatus and method for quantitative determination of target biomolecules.
  • Nanotubes such as nanotubes [References 9-1 1], semiconductor [References 12, ' 13], metal- oxide nanowires (NWs)[ Reference 14], and conducting polymer nanofilaments [Reference 15] have all been shown as capable of the label-free detection of small molecules, nucleic acids, and proteins.
  • SiNW Silicon nanowire
  • NW sensors detect the local change in charge density (and the accompanying change in local chemical potential) that characterizes a target/capture agent binding event. That changing chemical potential is detected as a 'gating' voltage by the NW, and so, at a given voltage, affects the source (S) ⁇ -> drain (D) current value, or I SD - However, that change is screened (via Debye screening) from the NW by the solution in which the sensing takes place [Reference 16].
  • Debye screening is a function of electrolyte concentration, and in a 0.14M electrolyte (which represents physiological environments such as serum) the screening length is about 1 nm [Reference 17]. Because of this, all reports on SiNW sensors for proteins or DNA have been carried out in low ionic strength solutions [References 12, 13 and 18]. .
  • a second challenge involves showing reproducible and high- throughput nanofabrication methods that can produce nearly identical NW sensors time and time again, and that allow for ⁇ multiple measurements to be executed in parallel.
  • Dimensional arguments [Reference 20] imply that that the fabrication of highly sensitive NW sensors requires non-traditional fabrication methods [References 21, 22].
  • all reports of NW sensors have utilized semiconductor NWs grown as bulk materials [Reference 23] using the vapor-liquid-solid (VLS) technique [Reference 24]. This method produces high quality NWs, but they are characterized by a distribution of lengths and diameters, and they also must be assembled into the appropriate device structure (or the device structure must be constructed around the nanowire [Reference 25]).
  • a third challenge involves the SiNW surface.
  • SiNWs for biomolecular sensing arises in part because of their high surface-to- volume ratio.
  • the native (1-2 nm thick) surface oxide on a SiNW may limit sensor performance due to the presence of interfacial electronic states [References 28, 29].
  • the oxide surface of SiNWs acts as a dielectric which can screen the NW from the chemical event to be sensed.
  • Covalent alkyl passivation of Si(IIl) surfaces can render those surfaces resistant to oxidation in air [Reference 30] and under oxidative potentials [Reference 31].
  • Recently, methyl passivated SiNWs were shown to exhibit improved field-effect transistor characteristics [Reference 32].
  • More complex molecules such as amine terminated alkyl groups, can be covalently attached to H-terminated Si surfaces (including SiNWs) via UV-initiated radical chemistry [References 33-36]. Such chemistry has been used for the' covalent attachment of DNA to VLS grown SiNWs [Reference 37]. DNA may also be immobilized on amine-terminated surfaces via electrostatic interactions.
  • SPR surface plasmon resonance
  • the molecule is often called the "target", while the complementary molecule is called the "probe".
  • the complement to a specific protein is a specific antibody (the probe)
  • the complement to a single-stranded oligomer of DNA is the complementary oligomer strand of DNA (the probe).
  • the probe molecule is attached to the surface of some substrate, such as a glass slide.
  • target molecules will bind to some fraction C of the target molecules in solution.
  • the rate at which the target biomolecules bind to the probe molecules is only determined by It 0n , kofl-and C. Ic 0n and k of rare typically independent of C.
  • SPR Surface Plasmon Resonance
  • a nanoelectronic device for detecting target molecules comprising: an array of nanowires serving as sensors of target molecules, the nanowires comprising i) electrically contacted regions at their ends, the electrically contacted regions being covered with an insulating material and ⁇ ) a central window region coated with a probe molecule; and a microfluidics channel placed across the array of silicon nanowires, the microfiuidics channel adapted to direct a flow of solution containing the target molecules.
  • a method for quantitatively determine a molar concentration of a target molecule comprising: providing an.
  • array of nanowires electrically contacting the nanowires at their ends; depositing an insulating layer over the nanowires; forming a window in the insulating layer along a region of the nanowires different from an electrically contacted region of the ' nanowires; treating the surface of the nanowires for later contact with probe molecules along the region different from an.
  • a method of fabricating a nanoelectronic device comprising: providing a silicon-on-insulator substrate; patterning a top silicon layer of the silicon-on-insulator substrate to obtain nanoscale wires; adding electrical contacts to the nanoscale wires: depositing an insulating layer on the nanoscale wires and the ' electrical contacts; and opening a window in the insulating layer to define a sensing area of the nanoscale wires.
  • a nanoelectronic device for detecting target molecules comprising: an array of nanoscale wires serving as sensors of target molecules; electrical contacts, electricaly contacting the nanowires at end regions of the nanoscale wires; an insulating material covering the end regions of the nanoscale wires and defining a window region of the nanoscale wires, the window . region of the nanoscale wires not being covered by the insulating material; and probe molecules, located on the nanoscale wires along the window region.
  • the present disclosure describes a nanoelectronic device that, when coupled with microfluidic devices, and operated in a certain fashion (described in the detailed description below), can measure Ic 0n and kotr for a particular target/probe combination, and C 5 the concentration of the target molecule.
  • the apparatus, methods and systems of the present disclosure can be extended to lower concentrations of target molecules and in particular of target biomolecules, that can be measured by competing techniques, and can thus be extended into clinically relevant concentration ranges of biomolecules.
  • the nanoelectronic device is- comprised of an array of nanowires (e.g., 5 to 10 silicon nanowires each of about 10 - 20 nm wide and a few micrometers long).
  • the nanowires serve as the sensors of the target bomolecules, and the doping level and nature of the dopant atoms within the nanowires determines the sensitivity limits and concentration ranges over which the nanowire sensors can operate.
  • the silicon nanowires are electrically contacted at either end, and the portion of the nanowires that are electrically contacted is covered with an insulating material. A microfluidics channel is then placed across the nanowires for directing the flow of solution containing the target biomolecules of interest.
  • the central region of the nanowires in between the electrical contacts is coated with the probe molecule.
  • a change in resistance is recorded by monitoring the electrical resistance of the nanowires. If measurements are done at two different values of target molecule concentration C, then the plots of time-dependent change in resistance can be utilized to determine Ic 0n and k o fr values for target/probe binding. Once ko n and k of r are known, then a solution containing the target biomolecule at an unknown concentration is introduced, and the concentration of the target molecule may be quantitatively determined.
  • the devices, methods and systems of the present disclosure are based on the ability of a single-stranded complementary oligonucleotide to significantly change the conductance of a group of 20 nm diameter SiNWs (p-doped at ⁇ 10 19 cm "3 ) in 0.165M solution by hybridizing to a primary DNA strand that has been electrostatically adsorbed onto an amine terminated organic monolayer atop the NWs.
  • This intimate contact of the primary strand with the amine groups of the NW surface brings the binding event close enough to the NW to be electronically detected.
  • the DNA hybridization is more efficient [References 10, 19].
  • the Superlattice Nanowire Pattern Transfer (SNAP) method [Reference 26] is used to produce highly aligned array of 400 SiNWs, each 20 nm wide and ⁇ 2 millimeters long.
  • Standard nano and microfabrication techniques are utilized to control the NW doping level [Reference 27], to section the NWs into several individual sensor arrays, to establish electrical contacts to the NW sensors, and to integrate each array into a microfluidic channel.
  • the resulting NWs exhibit excellent, controllable, and reproducible electrical characteristics from device-to- device and across fabrication runs.
  • the sensor platforms may also be fabricated in reasonably high throughput.
  • the NW sensors are doped so that their sensing dynamic range is optimized to match that of SPR for the detection of DNA hybridization.
  • the equivalence of these two methods, is shown and thus the use of SiNW sensors for quantitating analyte concentrations.
  • SiNW sensors can be optimized for significantly higher sensitivity than SPR, and thus can potentially be utilized to quantitate the concentrations of specific biomolecules at very low concentrations. That provides a unique application of these devices.
  • the applicants explore how the characteristics of SiNW sensors vary as the nature of the inorganic/organic interface is varied.
  • the applicants have found that SiNW sensors in which the native oxide • provides the interface for organic functional ization are significantly inferior in terms of both sensitivity and dynamic range when compared with SiNW sensors that are directly passivated with an alkyl monolayer.
  • Figure 1 shows a schematic representation of an array of nanowires according to an embodiment of the methods and systems herein disclosed.
  • Figure 2 shows a schematic representation of a side elevational view of the array of Figure 1 in a system according to an embodiment of the devices, methods and systems herein disclosed.
  • Figure 3 shows steps of a method to fabricate a nanoelectronic device in accordance with an embodiment of the present disclosure.
  • Figures 4A and 4B show a diagram ( Figure 4A) and a SEM image
  • Figure 4B of a single device section containing three groups of ⁇ 10 SiNWs in a microfluidics channel.
  • the wafer is covered with Si 3 N 4 except for an exposed active region or window with SiNWs.
  • the inset of Figure 4B shows a high resolution SEM image of 20 nm SiNWs.
  • Figure 5 shows two possible embodiments (Scheme 1 and Scheme 2) of the surface of the nanoscale wires in accordance with an embodiment of the present disclosure
  • Figure 6A shows an XPS (X-ray Photoelectron Spectroscopy) of a Si
  • Figure 6B shows Current- Voltage (IV) graphs of SiNWs functional ized by Scheme 1 of Figure 5 in solutions of varying pH.
  • Inset Solution gated (V S G) n-type hydroxy! terminated SiNW in solutions of varying pH.
  • Figure 7 shows a solution gating of SiNWs functionalized by Scheme 1 (light grey) and by Scheme 2 (dark gray) (V SD was 50 mV).
  • the right inset of Figure 7 shows IV curves of SiNWs in air with (black) and without (grey) oxide.
  • the left inset of Figure 7 shows resistances in air of SiNWs functionalized by Scheme 1 (left) and Scheme 2 (right).
  • Figure 8 shows a real-time response of SiNWs functionalized as in Scheme 1 to the addition of (a) lO ⁇ M ssDNA and (b) 10OnM complementary DNA.
  • the right top inset of Figure 8 shows a real-time SiNW response to the sequential addition of (a) 0.165M SSC, (b) 0.0165M SSC, and (c) 0.00165M SSC buffers.
  • the left inset of Figure 8 shows SPR (surface plasmon resonance) measurement showing the addition of 1 O ⁇ M ssDNA to poly-L-lysine coated CM5 sensor chip.
  • V SD 5OmV.
  • Figures 9A-9D show concentration-dependent, real-time sensing of complementary DNA by SiNWs and by SPR in 0.165M electrolyte.
  • Figure 9A shows real-time responses of SiNWs that were surface functionalized according to Scheme 1 of Figure 5 and coated with electrostatically adsorbed primary DNA.
  • the black trace represents exposure of the SiNW sensors to 100 nM non-complementary ssDNA.
  • Each curve represents measurements from a different set of NWs.
  • the inset of Figure 9A shows a fluorescence image of a Si(IOO) surface (with overlaying PDMS microfluidics chip) treated as in Scheme 1 of Figure 5 followed by lO ⁇ M primary DNA addition and addition of (microchannel a) 10OnM noncomplementary fluorescent DNA and (microchannel b) 10OnM complementary fluorescent DNA.
  • the PDMS microfluidic chip was removed before the image was collected.
  • Figure 9B is similar to Figure 9A, except the SiNWs were functionalized according to Scheme 2 of Figure 5.
  • the inset of Figure 9B is the same as the inset of Figure 9A, but the Si(IOO) surface was treated as in Scheme 2 of Figure
  • Figure 9C shows a SPR measurement of the hybridization of complementary DNA to electrostatically adsorbed primary DNA on a poly-L-lysine surface.
  • Figure 9D shows normalized SiNW responses for Figure 5 Scheme 1
  • Figure 10 shows a comparison of SPR-derived hybridization kinetic parameters with NW sensing data.
  • C I 0 nM. max
  • Figure 1 1 shows a schematic illustration of a method for the fabrication and assembly of a two-layer PDMS chip for solution injection (top) with a sensing device composed of SOI wafer and a single-layer PDMS chip with six separate microchannels (bottom)
  • Figure 1 is a schematic representation showing an array (10) of doped nanowires (e.g., silicon nanowires) coated with a probe biomolecule along a substantially central region (40) thereof.
  • the nanowires of the array (10) also comprise end regions (20, 30), electrically contacted to a first metal contact (50) and a second metal contact (60). Differently from the central regions (40), the end regions (20, 30) are covered with an insulating material.
  • Element (70) of Figure 1 shows a window (70) for the actual sensing area of the device and method in accordance with this disclosure.
  • the window (70) is the region of the nanowires not covered with an insulating material.
  • the region (70) will be exposed to the- solution that will later flow through the nanowires.
  • Coating (40) of the nanowires will occur inside the window (70).
  • the size of the window (70) and the extension of the coating (40) will define the size of the active sensing area.
  • the structure of the nanowire array (10) is defined by the GaAs/AlGaAs wafer grown by the MBE (Molecular Beam Epitaxy) technique, as also explained in other portions of the present disclosure.
  • the number of wires can be controlled by growing alternative layers of Ga As/A IGaAs. A possible number is 1400, and such number is just limited by the MBE.
  • Figure 2 shows a further schematic view, where the elements (10)-(70) previously described in Figure 1 are placed across a microfluidic channel (80).
  • the channel (80) will direct a flow (90) of solution containing the target molecules (e.g., biomolecules) of the present disclosure.
  • the detection mechanism according to the present disclosure is based on the charge of the target molecules. Therefore, any molecule or biomolecule that has a certain charge in the solution to be flown and proper capture agents (as later discussed in greater detail) can be flown.
  • target molecules can be DNA, RNA and proteins.
  • capture agent for non-bio molecules and those molecules have electrical charges, they can be used as targets, with a different surface chemistry.
  • the electrical resistance of the nanoscale wires is monitored. This is done in order to record change in resistance of the nanoscale wires over time at two different values of target molecule concentration to determine both an on-rate Ic 0n and an off-rate ko f ror target-probe binding. After this has .been done, a solution containing the target molecule at an unknown molar concentration is introduced, in order to quantitatively determine the molar concentration of the target molecule or biomolecule.
  • FIG. 3 shows steps of a method to fabricate the nanoelectronic device in accordance with the present disclosure.
  • a SOl (silicon-on- insulator) substrate (200) is provided, comprising silicon layers (210) sandwiching an insulator (e.g. SiO 2 ) layer (220).
  • nanowires (10) are made by etching the top silicon layer (210). The result of step S2 is shown both in cross-sectional and top view.
  • electrical contacts (50, 60) are made.
  • an insulating layer (230) e.g. silicon nitride, is deposited.
  • the insulating layer (230) is patterned to open a window (70) for the active sensing area of the nanowires (10).
  • Figure 4A shows a schematic perspective view on an embodiment of the present disclosure, where electrical contacts (50, 60) represent source/drain contacts of a transistor, as shown in' the enlarged inset of the Figure.
  • Figure 4 A also shows the microfluidics channel (80) and a platinum electrode (300) formed in a hole of the microfluidics channel (80) and connected to ground.
  • the platium electrode is used to ground the solution, in particular by setting the electrical potential to be identical to the ground of a lock-in amplifier used to measure the current and provide input signals.
  • This measuring arrangement is just one of many other measuring arrangements that can be devised for use with the present disclosure.
  • the two holes in the channel shown in the figure represent the inlet and the outlet of the microfluidic channel.
  • Figure 4B shows the embodiment of Fig. 4A in enlarged scale.
  • the rectangular aperture in the middle of Figure 4B shows the window opening (70), together with three sets of nanowires (10), each having first metal (source/drain) contacts (110) and second metal (source/drain) contacts (120).
  • One or more devices can be realized in a single embodiment.
  • Figure 4B shows three different stripes (and devices) in a single microfluidic channel. Electric contacts (150, 160) for a four-point measurement are also shown. Those contacts are intended to check how good the electrical contacts (110, 120) and are not intended to be used during sensing.
  • Figure 4B also shows darker regions (130, 140). Those regions show that the nanoscale wires of the three devices of Figure 4B can have different lengths and show nanowires covered by the insulating later, e.g., a silicon nitride layer.
  • the present disclosure shows how a quantitative, real time detection of single stranded oligonucleotides with silicon nanowires (SiNWs) in physiologically relevant electrolyte solution can be obtained.
  • Debye screening of the hybridization event is circumvented by utilizing electrostatically adsorbed primary DNA on an amine-terminated NW surface.
  • Two surface functional ization chemistries have been compared: an amine terminated siloxane monolayer on the native SiC> 2 surface of the SiNW (see Scheme 1 of Figure 5_), and an amine terminated alkyl monolayer grown directly on a hydrogen- terminated SiNW surface, as shown in Scheme 2 of Figure 5.
  • SiNWs without the native oxide exhibited improved solution-gated field-effect transistor characteristics and a significantly enhanced sensitivity to single stranded DNA detection, with an accompanying two orders of magnitude improvement in the dynamic range of sensing.
  • the applicants have developed a model for the detection of analyte by SiNW sensors and utilized such model to extract DNA binding kinetic parameters kon and k o pr. Those values have also been directly compared with values obtained by the standard method of surface plasmon resonance (SPR), and shown to be similar.
  • SPR surface plasmon resonance
  • the nanowires are characterized by higher detection sensitivity. The implication is that Si NWs can be utilized to quantitate the solution phase concentration of biomolecules at low concentrations.
  • This disclosure also shows the importance of surface chemistry for optimizing biomolecular sensing with silicon nanowires. Additional material suitable for the manufacturing of nanowires for the devices, methods and systems according to the present disclosure are identifiable by the skilled person upon reading of the present disclosure and will not be further described herein in detail.
  • FIG. 6A shows XPS scan in the Si/SiO x region.
  • the Si(IOO) surface with native oxide exhibited approximately 1.9 equivalent monolayers of SiO x .
  • the Si(IOO) surface treated according to Scheme 2 contained 0.08 equivalent monolayers of SiO x prior to TFA deprotection and 0.3 monolayers of SiO x after the deprotection step and a 10 hour exposure to Ix SSC buffer.
  • Scheme 1 -functionalized SiNWs shows a sensitivity to pH which is different than for native oxide-passivated NWs [Reference 45].
  • the isoelectric point of silica is ⁇ 2 [Reference 46], implying that for hydroxyl terminated, nonfunctionalized SiNWs at low pH, the SiOH groups are largely protonated.
  • negative charges on SiO ' should deplete carriers in the n-type SiNWs, causing a decrease in I DS (inset of Figure 6B). Above pH 4 the conductance is no longer modulated by increasing the pH, as most of the hydroxyl groups are deprotonated.
  • the surface is functionalized with amine (pK a ⁇ 9-10), the opposite trend is expected.
  • a hydrogen-terminated surface showed better sensitivity.
  • both of the above surfaces can be utilized.
  • the final goal of surface treatment for DNA sensing is that of making a positively charged surface, which can be done with different treatments and materials.
  • the applicants chose the amine because positively charged and widely used. Additional treatments of surfaces according to the present disclosure are identifiable by a skilled person and will not be further described herein in detail.
  • oxide covered SiNWs in ] ⁇ SSC buffer (0.165M, pH 7.2) respond weakly to the applied solution gate voltage, V SG , showing no significant on-off current transition between 0.8 and -0.8 Volts.
  • directly passivated SiNWs (Scheme 2 of Figure 5) exhibit on-off current ratios of ⁇ 10 2 .
  • Figure 7 strongly suggests that directly passivated SiNWs exhibit an enhanced response to surface charges and should therefore serve as superior NW sensors compared with similarly functional ized, but oxide-pass ivated SiNWs.
  • the Scheme 2 procedure does involve an HF etch step, which can be potentially detrimental to the device conductance.
  • Applicants thus checked the conductivity of SiNWs before and after photochemical treatment.
  • Lightly doped SiNWs provide for superior FET properties [Reference 47], and, in fact, Applicants have reported that lightly doped (10 17 cm “3 ) p- or n-type SiNWs are more sensitive biomolecular sensors than those discussed in the present disclosure [Reference 48].
  • Applicants' doping process preferentially dopes the top few nanometers of the SiNWs [Reference 49].
  • Figure 4 shows SiNW real-time detection of the electrostatic adsorption of 10 ⁇ M ssDNA, followed by the hybridization in Ix SSC buffer of 100 nM complementary DNA strand.
  • the resistance of p-type SiNWs is decreased with the addition of negative surface charges.
  • the metal contacts to NWs have been covered with Si ⁇ N 4 layer, and there is no background conductance through the solution.
  • Applicants have observed an insignificant change in the resistance of the NWs upon switching from dry environment to buffer solution (data not shown).
  • Figure 7 shows, changing the ionic strength of the solution does not affect the resistance.
  • the automated solution injection Figure H removes any baseline shifts or transient changes in the resistance when solutions are switched.
  • the surface density is approximately an order of magnitude higher than the average surface density of 10 12 cm “2 obtained when localizing biotinylated DNA on a streptavidin covered surface [Reference 52], Such high surface density of primary DNA is expected because the poIy-L-lysine treated surface is positively charged. It is likely that the amine-terminated SiNW surface has less surface charges than the poly-L-lysine covered surface and thus contains fewer sites for electrostatic adsorption of oligonucleotides.
  • Figures 9A-9D show real-time label free detection of ssDNA by SiNWs and by SPR.
  • the primary DNA strand was electrostatically immobilized on the sensor surface.
  • Known DNA concentrations were injected after a stable reading with Ix SSC buffer was obtained and the flow was maintained throughout the experiment. Different concentrations were detected with different groups of SiNWs.
  • Applicants observed that the hybridization on SiNWs is essentially irreversible on the relevant time scales when the analyte DNA was being washed away with the buffer solution. Such behavior is in contrast to SPR measurements, where the slow reversal of hybridization was observed (Figure 9C).
  • Figure 9D shows that the NW response ( ⁇ R/Ro) varies as log[DNA]. Such a logarithmic dependence has been previously reported [References 48, 53]. As shown in Figure 9D, the dynamic range of SiNWs is increased by about 100 after the removal of oxide and UV-initiated chemical passivation; the limit of detection (LOD) increased from about 1 nM to about 10 pM.
  • LOD limit of detection
  • SiNW sensors can be utilized to quantitate analyte concentration and binding constants.
  • the SiNW sensing response should be compared with other label-free, real-time methods such as SPR.
  • Experimental parameters should also be designed for both sensing modalities that are as similar as possible, as was described above.
  • applicants first discuss the use of electrostatically adsorbed primary DNA for detecting complementary DNA analyte.
  • Applicants then discuss the development of a self-consistent model that allows for the direct comparison of SPR measurements with nanowire sensing data.
  • the electrostatically adsorbed DNA coverage in applicants' SPR experiments was approximately 10 times higher, at 2.5 ⁇ lO 13 cm "2 . This difference in coverage likely arises from the differing methods of DNA immobilization; while in the applicant's embodiment the DNA is electrostatically adsorbed, other studies utilized a streptavidin-biotinylated DNA linkage for surface immobilization [References 19, 52], High surface coverage of primary DNA significantly reduces the efficiency of hybridization [References 51, 52].
  • the hybridized duplex of electrostatically adsorbed and covalently bound DNA may be structurally and energetically different.
  • ⁇ , surface density of bound analyte molecules
  • k m rate constant for association
  • k ojr rate constant for dissociation
  • C solution concentration of analyte (a constant under flowing conditions)
  • ⁇ max maximum number of binding sites available per surface area.
  • eq. 5 is expressing the fraction of bound analyte molecules at time t relative to the level at saturation in terms of ⁇ R (first term in brackets) and in terms of binding constants (second term in brackets). Time appears explicitly in the second term in brackets, while it is implicit in the first term in brackets (i.e., at a given time t there is a given R and AR ). If one plots the first term in brackets in eq. 5 (the term containing ⁇ R) against the second term in brackets (using k on and k o j values from an SPR analysis), one finds that the two curves are similar (Figure 10).
  • a second test of eq. 4 is to utilize it to extract binding kinetics. As one can infer from eq. 5, if eq. 4 is equivalent to the Langmuir binding model (eq. 2), then:
  • k on and A 0 J values can thus be extracted from measured resistance data.
  • R versus time traces can be selected at any two concentration values. Taking R and ⁇ R at an arbitrary point in time and noting ⁇ max (the resistance at saturation), two equations (one for each concentration C) and two unknowns are obtained. One can thus solve for k on and k oj f and compare directly with kinetic parameters obtained from SPR experiments.
  • the k on , hff > and KA values are summarized in Table 2.
  • the k on constants determined from the SiNW experiments are 3-5 times larger than k on obtained with SPR experiments.
  • the nanowire-measured k o f values are consistently quite close to those measured with SPR.
  • the variation in k on values may be a reflection of steric affects that arise from the unusually high surface density of primary DNA adsorbed onto the poly-L-lysine surfaces that were used for the SPR experiments [References 51, 52].
  • Table 2 shows kinetic parameters estimated from SiNW biosensors for the hybridization of 16-mer DNA and corresponding comparisons with analogous SPR and SPDS (surface plasmon diffraction sensor) [Reference 52].
  • the calculated concentrations (bottom row) were estimated with eq. 6, by using the pair of SiNW measurements that did not include the concentration to be determined. For example, the 1 nM and 100 nM measurements were used to determine the concentration at about 10 nM. Standard deviations are given in parentheses.
  • SiNWs concentration pair: (poly-L-lysine (avidin-bioti).
  • SiNWs with significantly reduced oxide coverage exhibited enhanced solution FET characteristics (Figure 7) when compared to SiNWs characterized by a native SiO 2 surface passivation.
  • Oxide covered, highly doped SiNWs were designed to exhibit a similar dynamic range of DNA detection as the best near-infrared imaging SPR technique [Reference 57].
  • - 1OnM for 18mer corresponding to ⁇ 10 u molecules/cm 2 .
  • the limit of detection was increased by two orders of magnitude, with an accompanying increase in the dynamic range.
  • the Si NW arrays were fabricated as previously described [Reference 39] and all fabrication was done within a class 1000 or class 100 clean room environment.
  • An embodiment of a NW sensor device employed in the present application for DNA sensing has been shown in Figures 4A and 4B.
  • the starting material for the SNAP process was an intrinsic, 320 A thick sUicon-on-insulator (SOI) substrate with (100) orientation (Ibis Technology Inc., Danvers, MA) and with a 1500 A buried oxide. Cleaned substrates were coated with either p-type (Boron A, Filmtronics, Inc.
  • SODs Spin-on-dopants
  • the SOD films were removed by brief immersion in piranha (70% H 2 SO 4 , 30% H 2 O 2 ), followed by a water rinse, and immersion in buffered oxide etchant (BOE; General Chemical, Parcippany, NJ).
  • BOE buffered oxide etchant
  • the SNAP method for NW array fabrication translates the atomic control achievable over the individual layer thicknesses within an MBE-grown GaAsZAl x Ga (I-X )As superlattice into an identical level of control over NW width, length and spacing. This method has been described in some detail elsewhere [References 26, 39] and will not be described here.
  • Applicants utilized the SNAP process to produce a 2 mm long array of 400 SiNWs, each of 20 nm width and patterned at 35 nm pitch (Fig 4B. inseti.
  • SiNWs were sectioned into -30 ⁇ m long segments using e-beam lithography (EBL) and SFe RlE etching, producing segments of ⁇ 10 SiNWs, each with a width of 20 nm.
  • EBL e-beam lithography
  • SFe RlE etching SFe RlE etching
  • the devices were annealed in 95% N 2 , 5% H 2 at 475 0 C for 5 minutes. This step greatly improves the characteristics of SNAP SiNW FETs.
  • Figure 4A To provide room for a . l cm by 1.5 cm PDMS chip with microchannels for analyte delivery to each section of the SiNWs ( Figure 4A). the electrical contacts were extended to the edges of the substrate using standard photolithography techniques followed by evaporation of 200 A Ti and 1500 A Au.
  • chromium was deposited over an active region of the NWs.
  • PECVD was used to deposit Si 3 N 4 film at 300 0 C (90OmT, 2OW, 13.5MHz) from N 2 (1960 seem), NH 3 (55 seem) and SiH 4 (40 seem) gases.
  • the nitride film was selectively etched with CHF 3 ZO 2 plasma over the protected NW region using PMMA as a mask, followed by the removal of chromium with CR-7C (Cyantek Corp., Fremont, CA).
  • microfluidics chip with six separate microchannels (Figure 11).
  • Such PDMS chip was fabricated using a standard photolithography: mixed PDMS (Dow Corning, Inc., Midland, MI) was applied over a pre-made photoresist molding on silicon wafer and incompletely cured at 8O 0 C for 30 minutes.
  • the chip containing microchannels was cut out of the PDMS layer and 0.5mm diameter holes were punctured to serve as microchannel inlets and outlets.
  • the fluidic chip and the device containing SiNWs were then brought into contact, with the lOO ⁇ m wide microchannels aligned over the individual nanowire sections.
  • the assembled device was cured to completion overnight at 8O 0 C;
  • a second PDMS chip which can sequentially inject four different solutions into one of six microchannels on silicon wafer.
  • sample injection chip is composed of two layers, control layer and flow layer ( Figure 11).
  • To fabricate the flow layer mixed PDMS was spin coated on a photoresist mold at 2500 rpm for 50 sec and incompletely cured at 80 0 C for 30 minutes.
  • Control layer was fabricated by applying mixed PDMS over a photoresist mold directly and incompletely curing at 8O 0 C, followed by the puncturing of holes for inlets and outlets. The two layers were aligned together and the inlets/outlets for the flow layer were created.
  • the two-layer PDMS chip was bonded to a glass slide utilizing an O 2 plasma treatment.
  • a sample injection chip applicants were able to control the injection and solution changing processes without disturbing the measurement, while maintaining the sensing device in an electrically isolated chamber at all times.
  • a waste outlet into the sample injection chip applicants were able to remove any bubbles arising from switching between different solutions, which also helped in maintaining a stable baseline reading.
  • the wafer was illuminated with UV (254 nm, 9 mW/cm 2 at 10 cm) for 3 hours. SiNWs were then rinsed in methylene chloride and methanol. The deprotection of t-Boc amine was carried out in a solution of TFA in methanol (1 :4 v/v) for 4 hours, followed by extensive methanol washing.
  • X-ray photoelectron spectroscopy was utilized to quantify the amount of oxide on Si (100) wafers after surface treatments outlined in Schemes 1 and 2. All XPS measurements were performed in an ultrahigh vacuum chamber of an M-probe surface . spectrometer that has been previously described [Reference 43]. Experiments were performed at room temperature, with 1486.6 eV X-ray from the Al Ka line and a 35° incident angle measured from the sample surface. ESCA-2000 software was used to collect the data. An approach described elsewhere [References 30, 43] was used to fit the Si 2p peaks and quantify the amount of surface SiO x , assuming that the oxide layer was very thin.
  • Complementary DNA was then immediately introduced and allowed to hybridize to the active surface.
  • the flow cell was regenerated with two 1 minute pulses of 50 mM NaOH, after which ssDNA was reabsorbed electrostatically before another cDNA pulse was introduced for hybridization.
  • a nanoelectronic device for detecting target molecules has an array of nanoscale wires serving as sensors of target molecules and electrical contacts, electrically contacting the nanowires at end regions of the nanoscale wires.
  • the end regions are covered with an insulating material.
  • the insulating material also defines a window region of the nanoscale wires, not covered by the insulating material.
  • Probe molecules are located on the nanoscale wires along the window region.
  • a microfluidic channel can also be provided, to allow flow of the target molecules.
  • a method of fabricating the nanoelectronic device is also shown and described.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Nanotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microelectronics & Electronic Packaging (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un dispositif nanoélectronique permettant la détection de molécules cibles. Le dispositif comprend un réseau de fils de d'échelle nanométrique servant de capteurs de molécules cibles et de contacts électriques, en contact électrique avec les nanofils à des zones d'extrémité des fils d'échelle nanométrique. Les zones d'extrémité sont recouvertes d'un matériau isolant. Le matériau isolant définit également une zone de fenêtre des fils d'échelle nanométrique, non recouverte du matériau isolant. Des molécules sondes sont situées sur les fils d'échelle nanométrique le long de la zone de fenêtre. Un canal microfluidique peut également être prévu, pour permettre l'écoulement des molécules cibles. L'invention concerne également un procédé de fabrication du dispositif nanoélectronique.
PCT/US2007/019166 2006-09-07 2007-08-29 Appareil et procédé de détermination quantitative de molécules cibles WO2008030395A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US84275006P 2006-09-07 2006-09-07
US60/842,750 2006-09-07

Publications (1)

Publication Number Publication Date
WO2008030395A1 true WO2008030395A1 (fr) 2008-03-13

Family

ID=39157551

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/019166 WO2008030395A1 (fr) 2006-09-07 2007-08-29 Appareil et procédé de détermination quantitative de molécules cibles

Country Status (1)

Country Link
WO (1) WO2008030395A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009013754A1 (fr) * 2007-07-24 2009-01-29 Technion Research And Development Foundation Ltd. Transistors à effet de champ à sensibilité chimique et utilisation de ceux-ci dans des dispositifs de nez électronique
WO2015059704A1 (fr) * 2013-10-22 2015-04-30 Ramot At Tel-Aviv University Ltd. Procédé et système de détection
CN110227565A (zh) * 2019-06-25 2019-09-13 京东方科技集团股份有限公司 微流控器件及制作方法、生物分子数量检测方法及系统
US10667750B2 (en) 2015-12-09 2020-06-02 Ramot At Tel-Aviv University Ltd. Method and system for sensing by modified nanostructure

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020117659A1 (en) * 2000-12-11 2002-08-29 Lieber Charles M. Nanosensors
US20050212531A1 (en) * 2004-03-23 2005-09-29 Hewlett-Packard Development Company, L.P. Intellectual Property Administration Fluid sensor and methods
US20050247961A1 (en) * 2004-03-09 2005-11-10 Chongwu Zhou Chemical sensor using semiconducting metal oxide nanowires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020117659A1 (en) * 2000-12-11 2002-08-29 Lieber Charles M. Nanosensors
US20050247961A1 (en) * 2004-03-09 2005-11-10 Chongwu Zhou Chemical sensor using semiconducting metal oxide nanowires
US20050212531A1 (en) * 2004-03-23 2005-09-29 Hewlett-Packard Development Company, L.P. Intellectual Property Administration Fluid sensor and methods

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YE Y. ET AL, ANALYTICA CHIMICA ACTA, vol. 568, no. 2006, 18 January 2006 (2006-01-18), pages 138 - 145 *
ZHANG Y. ET AL, PHYSICA B, vol. 382, no. 2006, 15 June 2006 (2006-06-15), pages 76 - 80 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009013754A1 (fr) * 2007-07-24 2009-01-29 Technion Research And Development Foundation Ltd. Transistors à effet de champ à sensibilité chimique et utilisation de ceux-ci dans des dispositifs de nez électronique
US10011481B2 (en) 2007-07-24 2018-07-03 Technion Research And Development Foundation Ltd. Chemically sensitive field effect transistors and uses thereof in electronic nose devices
US11243186B2 (en) 2007-07-24 2022-02-08 Technion Research And Development Foundation Ltd. Chemically sensitive field effect transistors and uses thereof in electronic nose devices
WO2015059704A1 (fr) * 2013-10-22 2015-04-30 Ramot At Tel-Aviv University Ltd. Procédé et système de détection
CN106164286A (zh) * 2013-10-22 2016-11-23 拉莫特特拉维夫大学有限公司 用于检测的方法和系统
JP2016535988A (ja) * 2013-10-22 2016-11-24 ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッドＲａｍｏｔ Ａｔ Ｔｅｌ Ａｖｉｖ Ｕｎｉｖｅｒｓｉｔｙ Ｌｔｄ． 検知のための方法及びシステム
US10274456B2 (en) 2013-10-22 2019-04-30 Ramot At Tel-Aviv University Ltd. Method and system for sensing
US10667750B2 (en) 2015-12-09 2020-06-02 Ramot At Tel-Aviv University Ltd. Method and system for sensing by modified nanostructure
US11275078B2 (en) 2015-12-09 2022-03-15 Ramot At Tel-Aviv University Ltd. Method and system for sensing
CN110227565A (zh) * 2019-06-25 2019-09-13 京东方科技集团股份有限公司 微流控器件及制作方法、生物分子数量检测方法及系统
CN110227565B (zh) * 2019-06-25 2021-03-19 京东方科技集团股份有限公司 微流控器件及制作方法、生物分子数量检测方法及系统
US11992836B2 (en) 2019-06-25 2024-05-28 Beijing Boe Technology Development Co., Ltd. Microfluidic device and method for manufacturing the same, and method and system for detecting the number of biomolecules

Similar Documents

Publication Publication Date Title
US20090066348A1 (en) Apparatus and method for quantitative determination of target molecules
Bunimovich et al. Quantitative real-time measurements of DNA hybridization with alkylated nonoxidized silicon nanowires in electrolyte solution
US8236595B2 (en) Nanowire sensor, nanowire sensor array and method of fabricating the same
EP1516174B1 (fr) Procede et dispositif de detection haute sensibilite de presence d'adn et d'autres sondes
CN103348238B (zh) 具有提高的灵敏度的纳米线场效应晶体管生物传感器
Vu et al. Fabrication and application of silicon nanowire transistor arrays for biomolecular detection
JP2012163578A (ja) ナノセンサプラットフォームを使用および構築する方法
US20150038378A1 (en) Biocompatible graphene sensor
US20080280776A1 (en) Method and apparatus for detection of molecules using a sensor array
US20180045717A1 (en) Systems and methods for single-molecule nucleic-acid assay platforms
US9434983B2 (en) Nano-sensor array
US20100273672A1 (en) Method and device for high sensitivity and quantitative detection of chemical/biological molecules
DE10221799A1 (de) Silicon-on-Insulator-Biosensor
Tsai et al. Surface potential variations on a silicon nanowire transistor in biomolecular modificationand detection
US20210396708A1 (en) Methods for detecting analytes using a graphene-based biological field-effect transistor
Ray et al. Label-free biomolecule detection in physiological solutions with enhanced sensitivity using graphene nanogrids FET biosensor
WO2008030395A1 (fr) Appareil et procédé de détermination quantitative de molécules cibles
Li et al. Effect of Electric Fields on Silicon-Based Monolayers
Midahuen et al. Optimum functionalization of Si nanowire FET for electrical detection of DNA hybridization
Mohanty et al. Field effect transistor nanosensor for breast cancer diagnostics
US20170088883A1 (en) Electronic platform for sensing and control of electrochemical reactions
US20210396709A1 (en) Method of manufacturing a graphene-based biological field-effect transistor
KR101032067B1 (ko) 차지펌핑을 이용한 바이오센서, 바이오센서 소자, 바이오센서 소자의 제조방법 및 바이오 물질 검출 방법
Thomas et al. Integration of silicon and printed electronics for rapid diagnostic disease biosensing
Abouzar Detection of molecular interactions using field-effect-based capacitive devices

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07811643

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07811643

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载