JP2006505806A - Electronic detection of nanotube-based biomolecules - Google Patents

Abstract

To detect protein-protein binding, nanoscale field effect transistor devices incorporating carbon nanotubes as conductive channels are used. The nanotube device is coated with an electron donating polymer and the receptor compound is bound to the polymer. The receptor compound is configured to bind to a specific biomolecule (one or more biomolecules). A device coated with a polymer coating and a receptor compound can be operated as a p-type field effect transducer. For example, exposure to biomolecules bound by a receptor significantly reduces the conductance at negative voltages, thereby reliably generating a response with an electronic signal.

Description

(Background of the invention)
(Field of Invention)
The present invention relates to the detection of biomolecules with nanotube-based sensors.

(Description of related technology)
Current biological detection methods are generally based on inherently complex optical detection principles, with many steps between the actual engagement of the analyte and signal generation, many types of reagents, many preparation steps, Requires signal amplification, complex data analysis, and / or a relatively large sample size.

  Nanowires and nanotubes are promising means for electron detection of chemical and biological species because of their small size, large surface area, and nearly one-dimensional electron transport (1). Field effect transistors (FETs) fabricated from component semiconducting single-walled carbon nanotubes (NT) have been extensively studied for their potential as sensors. Many characteristics of these devices have been clarified, and various mechanisms have been proposed to explain the sensing behavior of the devices. Devices incorporating carbon nanotubes have been found to be sensitive to various gases (e.g. oxygen and ammonia), and these findings have established the idea that such devices can operate as sensitive chemical sensors. It became so.

  Single-walled nanotube (“SWNT”) devices, including field effect transistors (“FETs”) and resistors, can be produced by chemical vapor deposition at 900 ° C. from iron-containing catalyst nanoparticles with a methane / hydrogen gas mixture. Can be produced using nanotubes grown on different substrates. Nanotubes can also be grown on the substrate using other catalytic materials and gas mixtures, and other electrode materials and nanostructures are described in U.S. Patent Application No. 10 / 099,664 and U.S. Patent Application No. 10 by Gabriel et al. No. 177,929, which are hereby incorporated by reference. At present, the technology for assembling practical nanostructured devices is still in the early stages. Although nanotube structures have shown promise for use as sensor devices and transistors, current technology is limited in many ways.

  For example, in order to detect biomolecules (such as proteins), it is desirable to use the small size and sensitivity of nanotube sensors and other nanostructure sensors. However, useful sensors of this type must respond selectively and reliably to specific types of molecular targets. For example, it may be desirable to selectively sense a particular protein without responding to the presence of other proteins in the sample. Examples of covalent chemical bonding of biomolecules, including proteins and DNA, to nanotubes are known in the art, but useful detection of specific proteins or other large biomolecules can be done in this way This has not been proven to be convincing. For one thing, covalent chemical bonds have the disadvantage of compromising the physical properties of the carbon nanotubes, and thus these types of structures are less useful as practical sensors. Furthermore, carbon nanotubes are hydrophobic and are usually non-selective in reaction with biomolecules.

Accordingly, it is desirable to provide a nanotube sensing device that is biocompatible and exhibits a high degree of sensitivity to a particular biomolecular target.
(Summary of Invention)
According to embodiments of the present invention, a nanotube sensor structure is provided that allows detection of protein-protein interactions and reduces or eliminates non-specific binding. The sensor can be operated as a nanostructured field effect transistor to detect the presence of a specific protein or other biomolecule. In addition, methods for manufacturing and operating sensing devices are provided.

  The nanostructured device of the present invention may include nanotubes (eg, carbon nanotubes) disposed along a substrate (eg, a silicon substrate). Nanotubes may span two conductor elements that serve as electrical terminals or as sources and drains. A passivation layer (e.g. of silicon monoxide) is deposited on the conductor elements and part of the nanotubes, leaving part of the nanotubes between the conductor elements exposed. Nanotubes can be coated with a thin polymer layer (eg, comprising poly (ethyleneimine) (“PEI”) and poly (ethylene glycol) (PEG)). In such a configuration, the device can be operated as an n-type FET (described in detail in US patent application Ser. No. 10 / 656,898). Advantageously, the polymer layer is hydrophilic and biocompatible, so the nanotube device is essentially non-reactive with large biomolecules such as proteins.

  A bioreceptor layer configured to react to specific biomolecules can be tied onto the polymer layer. For example, biotin is known to selectively bind to streptavidin. The bioreceptor layer must be configured to bind to the polymer layer. For example, a solution of biotin-N-hydroxysuccinimide ester reacts with a primary amine in PEI, thereby binding biotin molecules to the polymer layer. The bioreceptor layer may comprise a monolayer composed of separate bioreceptor molecules attached to a polymer layer.

  The device thus obtained exhibits a transconductance that varies depending on the presence of the target biomolecule in the sample environment. For example, a bioreceptor layer composed of bound biotin molecules selectively binds to streptavidin, thus reducing the transconductance at a negative gate voltage to a measurable extent. Therefore, this device can be used as a sensor for streptavidin. In order to detect other biomolecules, another bioreceptor layer configured to bind to the desired target can be provided in the device.

  A deeper understanding of the biomolecular sensor of the present invention, as well as the implementation of further advantages and objectives of the biomolecular sensor of the present invention, will be brought to those skilled in the art by considering the following detailed description of the preferred embodiments. Let's go. Reference is made to the accompanying drawings which are briefly described.

Detailed Description of Preferred Embodiments
The present invention provides a nanotube sensor that eliminates the limitations of the prior art for selectively detecting biomolecules. These advances have been demonstrated by the nanotube sensors of the present invention that have been shown to be selectively sensitive to the appropriately characterized ligand-receptor binding of biotin-streptavidin.

  In general, the present invention provides sensor structures that allow detection of protein-protein interactions and further reduce or eliminate non-specific binding. An essentially hydrophobic NT-FET coated with a hydrophilic polymer coating layer is used as the transducer. The hydrophilic nature of the polymer layer reduces the affinity of the nanotubes for non-specific protein binding (a hydrophobic environment is preferred for this binding). In the exemplary embodiment described below, biotin is covalently attached to the polymer. In use, the binding of bound biotin and complement protein streptavidin and the formation of a streptavidin-biotin complex can be detected electronically. Streptavidin-biotin complexes can serve as model systems for protein interactions, as has been extensively studied, and this binding is well understood. However, this does not limit the present invention.

FIG. 1 schematically shows a sensor 100 using carbon nanotubes 102 as a transducer. Nanotubes 102 are coated with a hydrophilic polymer coating 104, and bioreceptor molecules 106 are attached to the polymer coating by chemical bonds. Bioreceptor 106 can be selected to exhibit selectivity in binding to biomolecule target 107. Various receptor / target combinations are known, or such combinations can be found. In one embodiment, receptor 106 is biotin and target 107 is streptavidin. Additional bioreceptor molecules of the same or different type as molecules 106 can be further bound to polymer layer 104. Multiple types of such bioreceptor molecules (not shown) can be disposed on the surface of the polymer layer. Nanotube 102 can be coupled to source electrode 108 and drain electrode 110 on gate 112. A passivation layer 114 (eg, SiO ₂ ) known in the art may cover the gate substrate 112 (which may include silicon and other materials).

  Functionalization of the sensor structure through the polymer layer 104 has several advantages. First, polymers are used to bind the molecular receptor molecules to the nanotube sidewalls, thereby avoiding covalent chemical bonding of biomolecules to the nanotubes. Second, polymer coatings have been shown to change the properties of nanotube FET devices, and thus the coating process can be easily monitored. In particular, coating NTFET with polyethyleneimine (PEI) is advantageous because the device characteristics shift from p-type to n-type. Third, the polymer coating is useful in preventing nonspecific binding of proteins.

The effects of polymer coating, bioreceptor binding, and subsequent capture of biomolecules by the bioreceptor on the transconductance of the sensor device of the present invention can be understood as follows (however, this limits the present invention): Is never done). When coated with poly (ethyleneimine) (PEI), n-type doping occurs due to electron donating NH ₂ groups. PEI is not the only example of a polymer compound that can be used in such a method; other examples include poly (ethanolamine), poly (ethylene glycol), and polytetrahydrofuran bis (3-aminopropyl)- Examples include terminal polymers. The binding of bioreceptors (eg biotin) to PEI is due to covalent binding to the primary NH ₂ group, thus reducing the overall electron donating function of PEI and indicating the removal of electrons from the device. It is thought to cause a transconductance profile. Since only the primary NH ₂ site is involved in the binding with biotin, the p-type conductance observed before coating is not completely restored. It is reasonable to assume that when streptavidin-biotin binding occurs, there is a geometrical change that locally perturbs the coating, which reduces charge transfer efficiency and changes the transconductance of the device. It is worth noting that the functionalization of PEI layers or other polymer layers via primary NH ₂ groups can be applied to proteins as well as oligonucleotides.

  In addition to providing desirable electrical properties, layer 104, due to its hydrophilicity, reduces the affinity of nanotubes for protein binding, thereby increasing device selectivity. For biosensor and biomedical device applications, various polymer coatings and self-assembled monolayers are used to prevent the binding of undesirable chemical species to the surface, and these polymer coatings and monolayers are further Suitable for use with the present invention. Of the various polymers available for coating, poly (ethylene glycol) is one of the most effective materials and is widely used.

  An exemplary method 200 for fabricating a FET device similar to device 100 incorporating nanotubes as a conducting channel is shown in FIG. In step 202, a p-type NTFET is fabricated using nanotubes grown on silicon-doped 200 nm silicon dioxide by chemical vapor deposition (CVD) from iron nanoparticles with a methane / hydrogen gas mixture at 900 ° C. Can be manufactured. Conductors can be patterned from a 35 nm thick titanium film capped with a 5 nm thick gold layer, with a 0.5-0.75 μm gap between the source and drain on top of the nanotubes. Multiple nanotubes can be connected to the source and drain electrodes, at which time the properties of the individual tubes may change from metallic to semiconducting. Therefore, as a ratio between a range of device modulations ("on" source-drain current and "off" source-drain current (measured at -10V gate voltage and + 10V gate voltage, respectively)) Can be observed. Such devices show the behavior of p-type transistors before functionalization with appropriate polymer layers. A typical device resulting from the above process may have 0.5 μm wide paired conductors separated by a 0.5-0.75 μm gap, and these gaps are 10 μm long of a pair of conductors. It can be filled with 1 to about 5 nanotubes along. It goes without saying that many other arrangements are also suitable.

In step 204, device characteristics for the NTFET can be determined. As used herein, “device characteristic” refers to the gate voltage (V _g ) of the source-drain current (I _sd ) when measured at a gate voltage from +10 V to −10 V. _Represents the dependence as a function (I _sd (V _g )). Any other suitable method can be used to characterize the NTFET device. This device characteristic can be used as a baseline for subsequent calibration of the electrical response of the device.

  After the device characteristics are determined, a polymer functionalization layer can be deposited on the device in step 206. For example, the device can be placed in a 10% by weight aqueous solution of poly (ethyleneimine) (PEI, average molecular weight of about 25000, Aldrich) or a 10% by weight aqueous solution of poly (ethylene glycol) (PEG, average molecular weight 10,000, Aldrich) overnight. It can be dipped and then rinsed with water. Commercially available polyethyleneimine (PEI) can be used. The material is fairly branched and has a molecular weight of about 25000 and contains about 500 monomer residues. About 25% of the amino groups of PEI are primary, about 50% are secondary, and 25% are tertiary. After the coating process, the device must be coated with a thin layer of polymeric material (eg <10 nm). The final polymer coating can be observed by atomic force microscopy.

  In step 208, the desired biomolecular receptor can be bound to the polymer layer. If biotin is the desired receptor, the polymer-coated device can be biotinylated by immersion in a 15 mM DMF solution of biotin-N-hydroxysuccinimide ester (Sigma) at room temperature. This compound readily reacts with primary amines in PEI under ambient conditions, resulting in changes in device properties as described below. After soaking overnight, remove the device from the solution, rinse with DMF and deionized water, blow dry in a stream of nitrogen, and vacuum dry. FIG. 3A shows a chemical scheme that can bind biotin to a polymer coating. FIG. 3B shows a typical transconductance curve for a device coated with PEI before the biotinylation reaction and 1 hour and 18 hours after the reaction, respectively.

  Device properties can be examined as described herein after drying. Although the device of the present invention is also responsive in buffers and other liquids, the examples described herein function to show changes in device properties caused by various chemical and biological modifications. There must be. In a buffer environment, such a direct response can be somewhat obscured.

  The results according to the examples are described below. The biotinylated polymer-coated device prepared according to the above description was dried and then dissolved in a solution obtained by dissolving 2.5 μM streptavidin in 0.01 M phosphate buffered saline (pH 7.2, Sigma) at room temperature. At 15 minutes. The device was then rinsed thoroughly with deionized water and blown dry with nitrogen.

  An atomic force microscope (AFM) image of one of the devices after exposure to streptavidin labeled with gold nanoparticles showed the presence of streptavidin. The images show that streptavidin is effectively associated with the biotinylated PEI polymer coating on the nanotubes. From an image of a device consisting of about 800 nm long nanotubes and about 80 streptavidin molecules, it was speculated that they interacted directly with the nanotube conducting channel.

  The device characteristics of the sensor before chemical modification were p-type in the ambient environment (presumed to be due to exposure to oxygen). Coating the device with a mixture of PEI polymer and PEG polymer resulted in n-type device characteristics as shown in FIG. The electronic properties of the device after 18 hours of biotinylation are also shown in FIG. It should be noted that the p-type conductance observed before coating with PEI is not fully recovered after functionalization with biotin.

  The results of exposing the biotinylated polymer coated device to a streptavidin solution and the results of a control experiment (performed on another device) are shown in FIG. After exposure to streptavidin and subsequent streptavidin-biotin binding, it is clear that there is a significant decrease in source-drain current with respect to negative gate voltage and the device characteristics are negative or positive gate There is almost no shift towards voltage.

  Several control experiments were conducted to demonstrate the effectiveness of the device structure of the present invention in avoiding false positives and in detecting specific protein binding. First, an uncoated NTFET device was exposed to streptavidin. The change in device characteristics (as shown in FIG. 6) shows that streptavidin is associated with the device. However, it should be noted that in this case the most important result is a shift in device characteristics towards negative gate voltages. In contrast, if the device was polymer coated but not biotinylated, no change occurred upon exposure to streptavidin (shown in FIG. 7). This demonstrates the effectiveness of the polymer coating in preventing direct and non-specific interactions between streptavidin and nanotubes. Finally, even when streptavidin is bound in a state where the biotin binding site is blocked by complexation with excess biotin, the device properties of the biotinylated polymer-coated device change substantially as shown in Figure 8. There wasn't.

  Several conclusions can be drawn regarding the influence of biomolecules on the device electronics. First, exposure of a bare uncoated device to streptavidin shifts the transconductance towards a negative gate voltage, which makes the device less p-type, with little reduction in transconductance magnitude. This indicates that the main action of the nanotube-streptavidin bond is a charge transfer reaction with streptavidin that donates electrons to the nanotube. Biotin-streptavidin binding also has another effect of reducing current. At the same time, as shown in FIG. 5, the device characteristics change only for the negative gate voltage, and the transconductance in the positive gate voltage region is not affected.

  Interestingly, a similar effect can be observed in devices with charge carriers attached. This observed effect is due to the localization (delocalization) of the positively (negatively) charged ionic material by the negatively (positively) charged surface. Such a mechanism is also effective for the disclosed nanotube devices and may allow for electronic modification of biological reactions.

  With regard to the improvement of the NTFET device, the NTFET device can be further sensitive enough to achieve single protein detection and monitoring. As inferred from FIG. 5, the overall change in transconductance exceeds the noise level by about 10 times. According to the AFM image of the device, there are about 10 protein molecules very close to the carbon nanotubes. Combining these two numbers, we can deduce that there are 10 orders of streptavidin molecules from our current sensing level.

  From our experiments we can deduce almost the same detection sensitivity for uncoated nanotubes incubated with streptavidin (results for this are shown in Table 6). This result is in contrast to the relatively mild changes observed in devices where the active element is a nanowire (a channel with a substantially larger cross section).

  Thus, label-free electronic sensing, which incorporates a nanotube-based transducer as the central sensor element, can provide other features that are extremely useful in biomolecule sensing. Such sensors are small and responsive and require little power and therefore generate little heat. The active sensing area is sized to fit an individual protein or virus, and generally a small sample volume, when all current passes through the sensing point. Extremely high sensitivity. Importantly, the device can be specific for individual molecules and, in some cases, modulate the response to different molecules by using chemical and biological functionalization. be able to. It seems that it is generally more difficult to detect specific oligonucleotides directly, and the following findings are more useful than those for detecting individual proteins. Oligonucleotides in a sample generally exhibit a high degree of variation based on sequence, and the number of particularly important chemical species is often very small from two perspectives. This is because the sample may contain a fairly high concentration of a population of many oligonucleotide species that are very similar to one of the important chemical species.

  The principles and practice of the present invention are, over time, for developing cell-based electronic sensing (measuring the electronic response of biological systems) and for in vivo applications for cell physiology, medical screening, and diagnostics. It seems to contribute to the use of nanoscale devices. Sensor devices can be made according to the principles of the present invention and can generate a surface charge on a sensing element when a biomolecule is immobilized by applying a voltage across the sensor's elements. Such surface charges should interact with charged biomolecules, providing additional possibilities for the selective electronic detection of biomolecules, or the electrical manipulation of biological reactions at the molecular level. Therefore, the operation of the device of the present invention in such an aspect deserves further research.

  Having described a preferred embodiment of a nanotube sensor for selective detection of biomolecules and a method for manufacturing said nanotube sensor, it will be apparent to those skilled in the art that certain advantages of the present system have been achieved. is there. It should be understood that various modifications, adaptations, and alternative embodiments of the present invention are possible without departing from the scope and spirit of the present invention. For example, while a biotin-streptavidin device is disclosed, it should be understood that the concept of the present invention described above is for devices that utilize other receptor / biomolecule combinations. Is also applicable. The invention is further defined by the claims set forth below.

1 is a schematic diagram of a nanotube field effect transistor (NTFET) configured as a biomolecule sensor of the present invention. FIG. 3 is a flowchart showing typical steps of a method for manufacturing a nanotube biosensor of the present invention. 1 is a schematic diagram of a chemical scheme for binding biotin to a PEI / PEG polymer layer on a nanotube. FIG. FIG. 6 compares the transconductance of a native PEI / PEG-coated NTFET device with its transconductance after 1 hour and 18 hours of reaction with biotin-N-hydroxysuccinimide ester. FIG. 6 compares the transconductance of a bare NTFET, the transconductance of an NTFET coated with a PEI / PEG polymer layer, and the transconductance of an NTFET coated with a biotinylated PEI / PEG polymer layer. FIG. 3 compares the transconductance of NTFET devices coated with biotinylated PEI / PEG with and without streptavidin present. FIG. 6 compares the transconductance of a bare NTFET device with and without streptavidin. FIG. 3 compares the transconductance of a PEI / PEG coated NTFET device that does not contain a biotin receptor in the presence and absence of streptavidin. FIG. 3 compares the transconductance of biotinylated PEI / PEG coated NTFET devices with and without streptavidin complexed with biotin (which blocks the binding site of biotin).

Claims (15)

A nanotube coated with a hydrophilic polymer; and a molecular receptor compound associated with the hydrophilic polymer configured to bind to a biomolecule;
Including nanotube sensor.   2. The nanotube sensor according to claim 1, wherein the biomolecule is a protein.   2. The nanotube sensor according to claim 1, wherein the biomolecule is an oligonucleotide.   The nanotube sensor of claim 1, wherein the molecular receptor compound comprises a portion of a biomolecule.   2. The nanotube sensor of claim 1, wherein the molecular receptor compound comprises biotin.   2. The nanotube sensor of claim 1, further comprising a source electrode coupled to the nanotube and a drain electrode coupled to the nanotube.   7. The nanotube sensor of claim 6, further comprising a plurality of nanotubes coupled to the source electrode and a plurality of nanotubes coupled to the drain electrode. NTFET fabrication process;
Coating NTFET nanotubes with a hydrophilic polymer layer; and linking a bioreceptor compound to the hydrophilic polymer layer;
A method for producing a nanotube biosensor, comprising:   9. The manufacturing method according to claim 8, further comprising a step of determining device characteristics of the NTFET after the manufacturing step.   9. The method of claim 8, further comprising building a nanotube spanning counter electrode on the substrate.   9. The coating step further comprises a process selected from immersing the NTFET in a polymer solution; spin casting the polymer solution onto the NTFET; or drop casting the polymer solution onto the NTFET. The manufacturing method described.   9. The method of claim 8, wherein the step of binding further comprises covalently bonding the bioreceptor compound to the hydrophilic polymer layer.   9. The manufacturing method according to claim 8, wherein the binding step further includes binding molecules of the bioreceptor compound to amine groups of the hydrophilic polymer layer. A method of detecting a biological compound using a sensor comprising a nanostructured device coated with a hydrophilic polymer bound to an electron donating compound configured to bind to the biological compound comprising:
Exposing the nanostructured device to the sample; and signaling the detection of biomolecules based on transconductance values across the nanostructured device;
Including said method.   15. The method of claim 14, wherein the signaling step further comprises signaling a change in device characteristics.

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