Electronic detection of biological molecules using thin layers

Details Electronic detection of biological molecules using thin layers Electronic detection of biological molecules using thin layers

2001-10-02

2004-11-30

Fredman, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S006120, C435S091200, C536S023100, C205S777500

Reexamination Certificate

active

06824974

ABSTRACT:

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH AND DEVELOPMENT [Not Applicable]
FIELD OF THE INVENTION
This invention pertains to a biosensor for detecting and/or quantifying analytes. More particularly, this invention pertains to a biosensor based on a detection element that is a single macromolecule spanning two electrodes.
BACKGROUND OF THE INVENTION
Biosensors are devices that can detect and/or quantify analytes using known interactions between a targeted analyte and a binding agent that is typically a biological macromolecule such as an enzyme, receptor, nucleic acid, protein, lectin, or antibody. Biosensors have applications in virtually all areas of human endeavor. For example, biosensors have utility in fields as diverse as blood glucose monitoring for diabetics, the recognition of poisonous gas and/or explosives, the detection of chemicals commonly associated with spoiled or contaminated food, genetic screening, environmental testing, and the like.
Biosensors are commonly categorized according to two features, namely, the type of macromolecule utilized in the device and the means for detecting the contact between the binding agent and the targeted analyte. Major classes of biosensors include enzyme (or catalytic) biosensors, immunosensors and DNA biosensors.
Enzyme (or catalytic) biosensors typically utilize one or more enzymes as the macromolecule and take advantage of the complimentary shape of the selected enzyme and the targeted analyte. Enzymes are proteins that perform most of the catalytic work in biological systems and are known for highly specific catalysis. The shape and reactivity of a given enzyme limits its catalytic activity to a very small number of possible substrates. Enzyme biosensors rely on the specific chemical changes related to the enzyme/analyte interaction as the means for recognizing contact with the targeted analyte. For example, upon interaction with an analyte, an enzyme biosensor may generate electrons, a colored chromophore or a change in pH as the result of the relevant enzymatic reaction. Alternatively, upon interaction, with an analyte, an enzyme biosensor may cause a change in a fluorescent or chemiluminesceint signal that can be recorded by an appropriate detection system.
Immunosensors utilize antibodies as binding agents. Antibodies are protein molecules that generally do not perform catalytic reactions, but specifically bind to particular “target” molecules (antigens). Antibodies are quite specific in their interactions and, unlike most enzymes, they are capable of recognizing and selectively binding to very large bodies such as single cells. Thus, in addition to detection of small analytes, antibody-based biosensors allow for the identification of certain pathogens such as dangerous bacterial strains.
DNA biosensors typically utilize the complimentary nature of the DNA or RNA double-strands and are designed for the specific detection of particular nucleic acids. A DNA biosensor sensor generally uses a single-stranded DNA as the binding agent. The nucleic acid material in a given test sample is placed into contact with the binding agent under conditions where the biosensor DNA and the target nucleic acid analyte can form a hybrid duplex. If a nucleic acid in the test sample is complementary to a nucleic acid used in the biosensor, the two interact/bind. The interaction can be monitored by various means such as a change in mass at the sensor surface or the presence of a fluorescent or radioactive signal. In alternative arrangements, the target nucleic acid(s) are bound to the sensor and contacted with labeled probes to allow for identification of the sequence(s) of interest.
While the potential utility for biosensors is great and while hundreds of biosensors have been described in patents and in the literature, actual commercial use of biosensors remains limited. Aspects of biosensors that have limited their commercial acceptance include a lack the sensitivity and/or speed of detection necessary to accomplish certain tasks, problems with long term stability, difficulty miniaturizing the sensor, and the like. In addition, a number of biosensors must be pre-treated with salts and/or enzyme cofactors, a practice that is inefficient and bothersome.
SUMMARY OF THE INVENTION
This invention pertains to the development of a novel molecular sensing apparatus (biosensor) and to methods of use thereof. In preferred embodiments, the sensing apparatus comprises a first electrode, a second electrode, an insulator between the first electrode and the second electrode; and a binding agent (e.g. a biological macromolecule) connecting the first electrode and the second electrode. In particularly preferred embodiments, the binding agent is attached to the electrode in a manner that permits charge to flow from the electrode to the binding agent or from the binding agent to the electrode. Preferred binding agents include, but are not limited to, biological macromolecules (e.g. a nucleic acid, a protein, a polysaccharide, a lectin, a lipid, etc.) with a nucleic acid being most preferred. While the nucleic acid can be essentially any length, preferred nucleic acids range in length from about 5 nucleotides to about 5,000 nucleotides, more preferably from about 8 nucleotides to about 1,000 nucleotides or 500 nucleotides, still more preferably from about 10 nucleotides to about 300 nucleotides, and most preferably from about 15, 20, 25, 30 or 50 nucleotides to about 100 nucleotides or 150 nucleotides in length. Typically, the nucleic acid is of sufficient length to specifically hybridize to a target nucleic acid in a complex population of nucleic acids (e.g. total genomic DNA) under stringent conditions.
In preferred embodiments, the biological macromolecule is functionalized with a chemical group thereby facilitating the attachment of the macromolecule to the electrode(s). Preferred chemical groups include, but are not limited to a sulfate, a sulfhydryl, an amine, an aldehyde, a carboxylic acid, a phosphate, a phosphonate, an alkene, an alkyne, a hydroxyl group, a bromine, an iodine, a chlorine, a light-activatable (labile) group, a group activatable by an electric potential, and the like. In certain embodiments, the biological macromolecule is functionalized with a second biological macromolecule (e.g. a receptor, a receptor ligand, an antibody, an epitope, a nucleic acid, a lectin, a sugar, and the like). In preferred embodiments, however, such second biological macromolecules exclude nucleic acids.
Preferred insulators are insulators having a resistivity greater than about 10
−3
ohm-meters, more preferably greater than about 10
−2
ohm-meters, and most preferably greater than about 10
−1
, 1, or 10 ohm-meters. Suitable insulators include, but are not limited to SiO
2
, TiO
2
, ZrO
2
, quartz, porcelain, ceramic, polystyrene, Teflon (other high-resistivity plastics), an insulating oxide or sulfide of a transition metal in the periodic table of the elements, and the like.
In certain preferred embodiments, the first electrode and the second electrode are separated by a distance in the range of 1 to 10
10
Angstroms. Typically the first electrode and the second electrode are separated by a distance less than about 300 Angstroms, preferably less than about 150 Angstroms, more preferably less than about 70 Angstroms, and most preferably less than about 50 angstroms.
In certain embodiments, the first electrode and/or the second electrode have a resistivity of less than about 10
−2
ohm-meters, preferably less than about 10
−3
ohm-meters, more preferably less than about 10
−4
ohm-meters, and most preferably less than about 10
−5
, or 10
−6
ohm-meters. Particularly preferred electrodes comprise a material such as ruthenium, osmium, cobalt, rhodium, rubidium, lithium, sodium, potassium, vanadium, cesium, beryllium, magnesium, calcium, chromium, molybdenum, silicon, germanium, aluminum, iridium, nickel, palladium, platinum, iron, copper, titanium, tungsten, silver, gold, z

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