Biosensors have been and are being developed to detect, identify and quantify various biochemicals, ranging from proteins to toxins to RNA to c-DNA to oligos and to disease agents such as viruses, bacteria, spores and Prions. This list is by way of example, and is not intended to be complete. Some biosensors sense charge on the molecule. Many biochemicals carry a net charge. Electrophoresis methods and various blots exploit molecule net charge to affect physical separation of such molecules.
There is a significant problem with existing techniques such as electrophoresis and the various blots. These sensors are not specific in identifying the molecules in question unless significant post processing and labeling is employed. Further, a very large quantity of the tested biochemical is required for electrophoresis detection methodologies.
In many instances the number of molecules available for detection is very small and may be below the sensitivity threshold of the sensor, or may be problematic with respect to sensitivity. For example, some plasma proteins are of very low concentration. Toxins such as Botulinum toxin are notoriously hard to detect at lethal thresholds because of their very low lethal and sub-lethal, but still dangerous, concentrations. Mass spectroscopy requires a large number of molecules in order to achieve adequate detection sensitivity.
In the case of c-DNA and RNA sensing, the number of base pairs present may be low for adequate detection and determination of which one is trying to specifically identify. This is possible if, for example, only a few bacteria are present or the RNA is of low concentration because of function. Virus RNA may be of low density for samples monitoring air. Only a small portion of the RNA may provide the definitive identification signature. Overall this can lead to a relatively small amount of RNA or DNA actually involved in the definitive detection process, if only few bacterial or viruses are present.
In the case of proteins, the target molecule concentration may be very low in the sample. For example, with Prions (mad cow disease), if a fluid sample is taken from an animal's blood, the target protein concentration may be very low. With a rapid infection of humans, animals or plants with disease, the initial signature indicators may be present in only very small concentrations. For the very early stages of cancer, when one wishes to identify disease presence, definitive indicators may be present in only very small concentrations. An example includes the four or so proteins indicative of ovarian cancer. Where only small concentrations of target molecules are available, mass action effects can result in the bound target concentration being very low. A small percentage of the actual receptors, specific antibodies, available for bonding results in a very small detection signal, for example, as is the case of a lethal concentration of botulinum toxin. At the very earliest onset of disease, the density of indicative proteins, viruses, antibodies and bacteria may be very low, requiring putting a very high sensitivity burden on the sensing approach.
Sensors for the detection of target molecules using charge have been reported. The most commonly used to date are those using electrophoreses methods, such as the various blots. Semiconductor charge sensors have long been highly prized due to their compatibility with integrated circuits and attendant low cost manufacturing processes. An example is the ImmunoFET that uses a conventional MOSFET, absent a metal gate, and employing a reference electrode in solution.
Sensors sense a change in charge or chemical potential as a result of a chemical attachment to the gate region of the devices. Needs exist for sensitive sensors that can sense very low concentrations. Need exist for sensitive sensors that are IC compatible, especially CMOS compatible, and which overcome the obstacles reported for prior semiconductor based chemical sensors.
Contamination and pollution in water, air and foodstuff is a continuing threat to public health. Water contaminated with Pb, Hg, Dioxin, or other hazardous chemical substances is problematic. Air may be contaminated with hazardous chemicals, of which OSHA has a long list, either in the general environment, the home, the industrial workplace or the chemical factory. Food contamination is likewise problematic for public heath. The chemicals in question may be inorganic (such as Pb and Hg), organic (such as organic solvents) or biochemical such as viruses, bacteria, toxins and hazardous proteins.
Additional environmental threats arise from potential chemical use by terrorists. Such threats include the well-known toxins such as botulinum toxin and ricin, as well as many others. Another threat is that of explosives intentionally (such as bombs introduced by terrorists) or unintentionally (such as antipersonnel mines) found in some location.
There is a need for an electronic sensor that can detect such public health risk chemicals in water, air and foodstuffs. In general, such requirements include biosensors that may incorporate such specific chemical binding means as oligos, proteins and antibodies. These application sensors are discussed in a separate disclosure.
The invention provides chemical sensors for detecting environmental chemicals using surface and bulk selective chemical reactions.
Applying additional charge to the sensed molecule can provide additional detection signal. It is this need that is the object of the invention described herein. Such charge may be supplied in several non-obvious ways as described below.
Such attachment also may be used to enhance contact potential (chemical potential) changes on the gate arising from the attachment process. Additionally, the attachment may be used to provide additional confirmatory information on the specific target detection. These two applications will be discussed elsewhere.
Additional chemicals, large triangles in
In the present invention it is possible to bind very large charge complexes to the bound target (e.g., molecule or particle) and in this manner to provide ultrasensitive sensing detection of the original target.
The invention includes a molecule, molecular complex, particle or other structure that is attached to the target molecule (either bound to the sensor or to be bound to the sensors,
In particular embodiments, the sensing device may be affected by a change in the resistance of some key part of the device, C in
There are two general approaches to the charge amplification schemes:
In one approach, the sensor, prepared with a specific targeting receptor, is attached to the surface of the sensors, e.g., the gate layer shown in
In the second approach, the target species is mixed with a combination of specific chemical systems and particles and binds to those first, before binding to the sensor gate. Then the mix is exposed to the surface of the sensor, and portions of the already bound targets in solution bind to the gate surface, providing the sensor output detection signal. The charge amplification attachment occurs before binding to the sensor surface. Examples of such binding which provide added charge attachment are oligonucleotides and molecules, e.g., as with an antibody sandwich. Such systems can then bind to a receptor already attached to the surface of the sensor.
A wide range of combinations can be used. Particles such as nano particles or beads of polymer, metal, magnetic, coated and others may be used to bring large quantities of charge to the sensor surface through at least one binding event.
Gobs of material may be attached such as a gob of DNA, chroma cell material, proteins, or a gob of nanotube materials such as nanotubes fabricated from carbon or other chemicals.
There are many manufacturers of micro beads that are commonly used in the biochemical industries. These beads are often coated with a material that enhances the attachment of biochemicals of interest. The beads may be metal, polymer, semiconductor (such as Si or GaAs), or may be fabricated of other materials, and may include more than one material in a single bead. Such beads are routinely used to bind to proteins and nucleotide chemicals such as DNA, c-DNA, RNA, oligos, antibodies and other chemicals. Some beads have a polymer coated surface. Some beads carry a net charge on their own.
By attaching charged chemicals or particles to the beads, the beads are used as large charge suppliers that can, when attaching to the surface of a charge sensing device, deliver a substantial additional net charge to the gate, as shown in
This charge amplification is particularly important where the original target molecule is of low density, has low or no charge, and where, for example, binding to the receptors on the surface of the sensor is only partial. Some of the receptors are not bound. For very low concentrations of target chemicals, only a very small fraction of the receptors may be found. By way of example, for lethal concentrations of Botulinum toxin, only about 1 in 3000 antibodies are bound. Thus, while the original signal for the only sparsely bound target chemicals may be weak, the attachment of particles with substantial charge offsets and overcomes the problem of weak signals arising from limited binding events, and ultimately provides a large net charge for each target-binding event, and provides easy detection and identification of the original target.
Several illustrative examples are provided in
The current invention employs the concept of chemical reaction with a pre-selected surface integrated with a suitable semiconductor sensor devices as schematically represented in
The material layer M may be an elemental material, an organic material, a biochemical material, a polymer material or other material. The material in the environment that reacts with material M may be elemental (such as Pb), organic (such as an insecticide), biochemical (such as a protein or toxin), a spore, a nerve agent, an explosive compound's vapor, or a combination of agents, by way of example.
In addition to polymer, biochemical, compound or elemental material coatings, membranes may also be adhered to the surface. Such membranes can react with chemicals present as well as transmit chemicals to an underlying surface.
The invention includes a semiconductor conducting region integrated with various semiconductor device structures such as the one shown in FIGS. 8A-D, by way of example. In
The reactant R may be a thick coating (such as iron oxide) or a monolayer, or a fraction of a monolayer. The degree of coverage of the monolayer provides a signal via the change in the underlying conductor C, as shown in
By way of example,
Similar changes occur when charge is associated with the new reactant R.
Design of the device for the maximum sensitivity to a contact potential change may include the use of a very thin insulation region I. Other design features may be selected to maximize the sensitivity or affect a pre-selected sensitivity features such as sensitivity range through various design considerations that are suitable for the device structure selected for sensing applications.
Capacitive sensing is also explained. Measuring capacitance may be used to monitor the devices, such as illustrated in
In general terms, applications include, but are not limited to detection and quantification of an environment chemical in air, water or incorporated into a food supply. Specific applications, by way of example, but not limited to these examples, include the detection of, or alteration of: corrosion characterization, characterizing chemical coatings (such as for protection), detection of target chemicals (such as explosive vapors, insecticides, corrosive chemicals, Pb, Hg, and many others, organic solvents, inorganic materials in general, hazardous compounds or elements, insecticides (and nerve gas), biochemical materials, polymers, gases, fluids, or coatings.
For example, using the invention, one can characterize the quality of a coating for protection against corrosion, such as in seawater. Alternatively, one can use the device to detect the presence of a compound, such as explosives or insecticides. Still another application is to measure the contact potential of materials used in the integrated circuit industry in order to integrate such information into the design of the integrated circuit. For example, Au and Al have different influences. Other more exotic materials have different influences. By way of further example, the top material region may comprise a collection of nano tubes of carbon or some other material. Many other applications will be obvious to those of skill in the chemistry art upon reading this disclosure or hearing a description equivalent to this disclosure or a part of this disclosure.
The invention has many application regimes such a monitoring of water quality, air quality and inspecting for explosives at airports.
This invention includes multiple applications CMOS compatible sensors, distributed channel bipolar devices (DCBDs), biosensors, force sensors, magnetic sensors and optical sensors.
These and further and other objects and features of the invention are apparent in the disclosure, which includes the above and ongoing written specification, with the claims and the drawings.
A bio-bar code assay method is described in the following example. “A” is probe design and preparation. “B” is PSA detection and bar code DNA amplification and identification. In a typical PSA-detection experiment, an aqueous dispersion of MMP probes functionalized with mAbs to PSA (50 μl of 3 mg/ml magnetic probe solution) is mixed with an aqueous solution of free PSA (10 μl of PSA) and stirred at 37° C. for 30 min (Step 1). A 1.5-ml tube containing the assay solution is placed in a micro-centrifuge tube separator at room temperature. After 15 s, the MMP-PSA hybrids are concentrated on the wall of the tube. The supernatant, solution of unbound PSA molecules, is removed, and the MMPs are re-suspended in 50 μl of 0.1 M phosphate-buffered saline (PBS) (repeated twice). The NP probes (for 13-nm NP probes, 50 μl at 1 nM; for 30-nm NP probes, 50 μl at 200 pM), functionalized with polyclonal Abs to PSA and hybridized bar-code DNA strands, are then added to the assay solution. The NPs react with the PSA immobilized on the MMPs and provide DNA strands for signal amplification and protein identification (Step 2). This solution is vigorously stirred at 37° C. for 30 min. The MMPs are then washed with 0.1 M PBS with a magnetic separator to isolate the magnetic particles. This step is repeated four times, each time for 1 min, to remove everything but the MMPs, along with the PSA-bound NP probes. After the final wash step, the MMP probes are re-suspended in NANO pure water (50 μl) for 2 min to dehybridize bar code DNA strands from the nanoparticle probe surface. Dehybridized bar code DNA is then easily separated and collected from the probes with the use of the magnetic separator (Step 3). For bar code DNA amplification (Step 4), isolated bar code DNA is added to a PCR reaction mixture (20-μl final volume) containing the appropriate primers, and the solution is then thermally cycled (20). The bar code DNA amplicon is stained with ethidium bromide and mixed with gel-loading dye (20). Gel electrophoresis or scanometric DNA detection (24) is then performed to determine whether amplification has taken place. Primer amplification is ruled out with appropriate control experiments (20). Notice that the number of bound NP probes for each PSA is unknown and will depend upon target protein concentration.
In the present invention, an approach is used by incorporating a similar general product generating system with binding antibodies and a product with the oligonucleotides attached to the beads. In the present invention, the latter beads are then subsequently being bound instead to the surface of the sensor. For the current invention, one uses a bead preparation method with the beads modified to bind the beads with heavy nucleotide attachment to the sensor and thereby to add the significant charge of the oligo nucleotides on the bead to the net charge on the sensor gate, shown in
An antibody (Y) is used, by way of example, to bind an antigen to the sensor's gate surface. An identical antibody is included in the mix of compounds attached to the bead and binds to the exposed antigen forming a sandwich. An additional second class of compounds is represented by the box square shaped receptor also attached to the bead. This later compound may be any other suitable chemical such as, for example, an antibody, DNA, c-DNA, RNA, an oligo, a protein or other chemical or chemical system specific binding system. The bead carries substantial additional charge already, but this can be increased as shown in
In
By way of example, this could be another antigen specific to a (square) antibody, or could be a c-DNA specific to a (square) oligo, or a protein system, or other chemical specific pair system. In this way, exposure to the new chemicals, squares in
The attachment systems may be used to incorporate complementary targets that add confirmatory information or redundant information on the sensor target. The system may also be used for subsequent processing for other targets that may be present, with receptors or recognition elements to the additional target(s) provided on the attaching components. For example, an attaching bead may have additional receptors (recognition elements) for detecting subsequent cofactor or other target molecules. Confirmatory information provides increased confidence in the target identification.
The above are change amplification schemes that are presented by way of example. The invention in a more general form is represented in
It is also noted that the resistive, i.e. conductive, region C of
Cascading of bead binding may be achieved to further increase charge amplification.
It is noted that some beads may carry net charged and these may be used in the invention, or may be prepared to carry charge through coating, chemical treatment or other means.
DNA, Oligo and RNA charge amplification methods may be used. Nucleotide chemicals may be included in the charge sensing amplification as indicated above and in the attached figures.
Examples of attached charge systems are discussed above and include proteins, nucleotides, beads, antibodies, many biochemicals and charged particles, such as metal beads that have been charged.
Protein amplification methods are described. It is well known that proteins carry charge. Proteins and/or protein chains may be added to the charge amplifying components, such as to another protein, an antibody, a stand of RNA or DNA, or to a bead, by way of example, to provide charge increase at the gate region and on the component particles used in the invention.
Antibody-DNA charge amplification is discussed. Examples are in the figures and discussed above to show how antibodies may be incorporated into the gate charge amplifying schemes.
Rolling circle amplification, as discussed herein, may be used to increase the length of a nucleotide chain, and thus the amount of charge it is providing. The rolling circle DNA chain may be attached to antibodies or other particles.
Detergents can carry charge. Coating beads of surfaces or biochemicals with such detergents can thus add charge.
It is well known that pH affects the charge on biochemicals. The pH of the test solution may be changed to enhance charge and also to provide confirmatory information.
PCR enhancement is discussed. Where nucleotide chemicals are incorporated, PCR may be employed. PCR may be used to further increase the amount of pre-selected nucleotides that are selectively incorporated to further increase sensing sensitivity. Other nucleotide amplification means, such as strand displacement amplification may also be used. The invention disclosure extends to all such amplifying schemes to be embraced in the current invention.
Other methods of increasing the gate charge will become obvious to those of skill in the art on reading this disclosure or learning of the invention. And, these too are claimed as a part of the invention.
Applications of the sensors and the charge amplification schemes are extensive. By way of example, some of the applications and markets for the invention include: Proteomics, disease diagnostics (human, animal, plant), drug discovery, co-factors, confirmation testing, genetics, toxin arrays, spores, cancer, drug efficacy, blood banking, arrays incorporating addressing redundancy, confirmation and multiple targets, and others.
It is noted that some of the approaches described in this document may also be applied to chemical potential enhancement where the sensor is targeting a chemical potential of materials attached to the gate region. By way of example, selected metal beads may be employed.
While the invention has been described with reference to specific embodiments, modifications and variations of the invention may be constructed without departing from the scope of the invention, which is defined in the following claims.
This application claims the benefit of U.S. Provisional Application No. 60/554,610, filed Mar. 18, 2004, U.S. Provisional Application No. 60/554,612, filed Mar. 18, 2004, and U.S. Provisional Application No. 60/554,616, filed Mar. 18, 2004, which are hereby incorporated by reference in their entirety.
Number | Date | Country | |
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60554610 | Mar 2004 | US | |
60554612 | Mar 2004 | US | |
60554616 | Mar 2004 | US |