Field of the Invention The present invention relates generally to the fields of sensor technology in molecular diagnostics. More particularly, it concerns the electrochemical detection of nucleic acid segments of nucleic acids.

The effects of pathogenic organisms cause closely related human and plant problems.

Molecular controls of both are becoming possible as a result of technological advances in the area of DNA diagnostics. When scientists can identify genes that are unique to a stage of growth of a particular parasite, bacterium or virus, drugs can be targeted to these genes.

Similarly, knowledge of the products of these genes can be incorporated into the design of improved treatments against human or plant pathogens. Advances in molecular biology have led to a rapidly increasing store of knowledge on the genomes of disease-causing organisms.

This knowledge can potentially be used to identify nucleic acid sequences that are specific to pathogens, to locate therapeutic targets, and to develop new and improved drugs to treat or prevent disease.

Human health problems and problems requiring better pest control in agriculture can benefit from use of DNA technologies in diagnostics. The increasing worldwide problems of disease caused by parasites, bacteria and viruses has been reviewed (www. niaid. nih. gov !. The emergence of drug-resistant strains, a breakdown of public services, changes in climate, greatly increased population mobility, a lack of understanding of the threat, and a focus on other diseases, such as cancer, have all led to the resurgence of disease-causing organisms all over the world at a time when infectious diseases were thought by many of us to be controlled.

Moreover, newly emerging diseases, drug-resistant diseases, insecticide-resistant carriers of disease, and the growth of disease-susceptible human and plant populations all pose an enormous challenge through the next century to improve diagnosis, treatment, and prevention of

diseases associated with pathogenic organisms. As a result of the spread of drug-resistant parasites and insecticide-resistant mosquitoes, our tools for dealing with this threat are diminishing at the same time that the threat is increasing.

The determination of a specific DNA or RNA target nucleic acid sequence or segment present in air, food, water, environmental or clinical samples is consequently of great significance in the medical microbiology, food and water safety-testing and environmental monitoring fields. The detection of the presence of a DNA or RNA sequence in a sample can rapidly and unambiguously identify bacterial, viral or parasitic agents of concern. Diagnosis of numerous infectious and inherited human diseases can be done with clinical assays that detect known DNA sequences characteristic of a particular disease.

Recent advances in molecular diagnostics have naturally focused on methods of detection at the genetic level. Since the advent of PCRTM technology, the ability to detect point mutations, allelic variation, the presence of minute amounts of a pathogen and identify species or individuals from microscopic samples, to give a few examples, has been vastly improved.

Yet even with the advances of PCRT" technology many limitations still exist which prevent diagnostic assays from being as versatile as desired or needed.

Unfortunately, few detection methods are suitable for routine diagnostic use either in the clinical laboratory or in the field setting. Many assays are not sufficiently rapid, inexpensive, simple or robust for routine application. In recent years, DNA assays that do not require radioactive labels have been developed for the research and clinical markets to eliminate hazards of handling radioactive materials. The most common non-radioactive labels in use today are fluorescent, chemiluminescent, or enzyme types. Several companies have developed automated instrument/assay systems based on non-radioactive labels, but realizing the necessary sensitivity and high throughput has required complicated techniques and expensive equipment, such as large laser-driven scanners used in the analysis of assays using gels, blots, and current- generation DNA chips.

One of the biggest problems, for example, with highly sensitive assays, such as PCRT"" based assays, is contamination, such as by an extraneous air-borne DNA or by human contact.

In general, if a molecular diagnostic assay is highly sensitive and can detect minute quantities of a selected or target nucleic acid segment then the sample to be assayed must be highly purified

or at least not contain extraneous nucleic acid fragments which may be at least partially complementary to the target nucleic acid segment. Otherwise false positive or ambiguous readings can result. Of course, obtaining highly purified samples can be cost ineffective and time and labor intensive. Strong technical expertise and well-equipped diagnostic laboratories are required in most cases. Thus in many instances where a highly sensitive assay is desirable, it is impractical, if not impossible, to perform such assays.

Further, if it is desirable to detect more than one target nucleic acid species in a sample, or if the sample is highly complex, then highly sensitive assays must be refined to detect the desired targets. Although it is not entirely understood, it is well known that many highly sensitive assays suffer from undue interference caused by background sample material. In fact, the time and labor required to refine some assays is so great that the assays are not useful and less sensitive means of analysis must be employed.

Diagnostic assays that are less sensitive are less susceptible to contamination problems and usually require less pure samples but cannot detect minute amounts of target. Thus larger or more concentrated samples must be obtained. In some cases, it is impossible to obtain more sample and in other cases obtaining more sample can be time, labor and cost consuming.

Probe assays, including oligonucleotide-probe and gene-probe assays, have been developed recently in an attempt to take advantage of the ability to detect DNA or RNA sequences characteristic of specific bacteria or viruses with high sensitivity and replace conventional detection methods. However, these techniques still tend to be labor-intensive and often require significant technical training and expertise. Further, highly sensitive gene-probe assays still require specialized equipment and are generally not compatible with field settings.

Those that can be used in a field setting usually are limited to determining the presence or absence of a target nucleic acid fragment and cannot meaningfully quantitate it. Thus the utility of gene-probe assays for environmental monitoring and other uses outside of a laboratory setting is limited. Detection assays which can provide a qualitative and quantitative early warning of the presence of pathogens, infectious organisms and parasites harmful to human and environmental health are still needed.

In addition, gene-probe assays often use a label that is either toxic or requires substantial expertise and labor to use. Radio-labeling is one of the most commonly used techniques

because of the high sensitivity of radio-labels. But the use of radio-labele'es is expensive and requires complex, time consuming, sample preparation and analysis and special disposal.

Alternatives to radioactivity for labeling probes include chemiluminescence, fluorimetric and colorimetric labels but each alternative has distinct disadvantages. Colorimetry is relatively insensitive and has limited utility where minute amounts of sample can be obtained. Samples must also be optically transparent. Fluorimetry requires relatively sophisticated equipment and procedures not readily adapted to routine use. Chemiluminescence, although versatile and sensitive when used for Southern blots, northern blots, colony/plaques lift, DNA foot-printing and nucleic acid sequencing, is expensive, and thus is not suitable for routine analysis in the clinical laboratory.

Another limitation to the versatility of oligonucleotide-probe assays is that virtually all current oligonucleotide-probes are designed as heterogeneous assays, i. e., a solid phase support is used to immobilize the target nucleic acid so that free, non-hybridized probe can be removed by washing. Complex procedures and long incubation times (one to several days) are usually required, which makes these assays difficult to incorporate into the simple and rapid formats desirable for clinical applications or on-site analysis.

Alternatives to gene-probe and other assay methods of detecting nucleic acid sequences have employed electrochemical biosensors that employ intercalators and discriminate between immobilized single-stranded and double-stranded DNA. While such biosensors are capable of detecting a known target DNA sequence, they are handicapped by the fact that the electrode must be cleaned between each use. The procedures used to strip away the hybridized target DNA from the electrode surface are not suitable for widespread screening applications, such as clinical diagnostics where labor and expense must be kept minimal and speed is essential, or in settings outside of the laboratory such as the field testing.

One problem that is particularly acute in field use is the general lack of suitable equipment. Most assay equipment is large, complex, and fragile. Equipment for field use typically must be compact, simple, and rugged. For instance, field equipment must be small enough to be easily handled, and should preferably be hand-held. Ruggedness is desirable so that the equipment can survive shock, vibration, and temperature extremes much more severe than any found in typical laboratory environments. Note also that a rapid assay is particularly

important in this setting because of a higher probability of environmental factors vitiating the test.

Thus, there is a need for improved detection of nucleic acid sequences that may be identified with specific pathogens. There is also a need for more rapid, less labor intensive and cost effective clinical assays for the detection and identification of diseases and disorders affecting mankind as well as field portable assays and kits which can be used to monitor the sources of such infections. Unfortunately, few assays are currently available for routine monitoring and/or diagnostic use because of the expense, complexity and/or physical limitations which prevent their use outside of a well-equipped laboratory. As discussed, the few assays that do exist have limited applications and do not meet the diverse needs of clinical diagnostics and field testing.

The present invention is directed to resolving one or all of the problems mentioned above.

The invention, in its various aspects and embodiments, is a printed circuit board biosensor and a use for the same. The printed circuit board biosensor comprises a printed circuit board and a bioreporter. The printed circuit board includes a working electrode and a reference electrode formed thereon. The bioreporter is operably linked to the working electrode and capable of generating an electrochemical signal upon specifically recognizing a target molecule to be detected in a sample when subjected to an electrical potential applied across the working and reference electrodes. The printed circuit board biosensor may, in some embodiments, comprise part of a system for detecting a target molecule in a sample. Such a system might include, in addition to the biosensor, means for detecting the electrochemical signal when an potential is applied across at least one reference electrode and at least one working electrode and/or means for applying the electrical potential. The printed circuit board, and systems including the same, may also comprise kits when sold with instructions on their use in accordance with the present invention.

The invention may be understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which: Figure 1 illustrates an apparatus constructed and operated in accordance with one aspect of the invention.

Figure 2 is a block diagram of the electronics for one particular embodiment of the monitor in Figure 1; Figure 3 depicts one particular embodiment of the sensor in Figure 1, in which at least a reference electrode and a working electrode are printed on a printed-circuit-board substrate, in an elevational view; Figures 4A-4F illustrate several variations on the particular embodiment of the sensor in Figures 1 and 3 in a series of plan views; Figure 5 is a flow chart of the basic operation of one particular embodiment of the monitor in Figure 1; Figure 6 illustrates electrical potentials applied across the electrodes of the sensor in Figures 1 and 3 using three variations of Pulsed Electrochemical Detection ("PED") as may be used in various, alternative embodiments of the present invention; Figures 7A-7B schematically diagrams the configuration of probes and labels for one particular embodiment of the present invention; and Figures 8A-8C illustrate the human interaction with the apparatus of the invention in the testing of a sample.

While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description herein of specific embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

Illustrative embodiments of the invention are described below. In the interest of clarity, not all features of an actual implementation are described in this specification. It will of course be appreciated that in the development of any such actual embodiment, numerous implementa-

tion-specific decisions must be made to achieve the developers'specific goals, such as compli- ance with system-related and business-related constraints, which will vary from one implementation to another. Moreover, it will be appreciated that such a development effort, even if complex and time-consuming, would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

The present invention provides, apparatus, methods, and compositions for the selective, rapid, and sensitive electrochemical detection of nucleic acids such as are found in microbes, including bacteria, viruses and parasites. These nucleic acids may be present in environmental samples, such as food, air, fresh and/or marine waters, or in biological samples, such as tumor cells and other cells exhibiting abnormalities. Practically any nucleic acid may be detected so long as its sequence is known or sufficiently identified to allow selection of appropriate hybridization probes. The methods are applicable to the analysis of samples in clinical, research, or field settings and are also useful for monitoring in genetic epidemiology and environmental remediation.

The Apparatus of the Invention Turning now to Figure 1, the invention provides an electrochemical detection system 100 that utilizes probes (not shown in Figure 1) and at least one printed circuit board, biosensor 110 for the specific detection of target-DNA sequences at the surface of a working electrode of an electrochemical sensor. The sensors 110 are read in a manner discussed more fully below by a monitor 112. Homogeneous electrochemical assays are provided, i. e., assays that utilize a single-tube format-such that a solid phase is not required to immobilize a target-DNA segment or a hybridized target-DNA segment/probe in order for detection to occur.

Construction of Apparatus The Monitor. The monitor 112 may be any suitable monitor known to the art. In the particular embodiment illustrated, the monitor 112 is an electrochemical monitor available from: AndCare, Inc.

P. O. Box 14566 Research Triangle Park, NC 27709 Phone: (919) 544-8220 Fax: (919) 544-9808 www. andcare. com

This electrochemical monitor is sold under the mark Pulse Amperometric Monitor (Version 5.1) and operates on separately available software. These portable electrochemical monitors are desirable because they: are inexpensive, hand-held potentiostats; may be powered with a 9V battery or AC power supply; include two modes of operation: Staircase and Square Wave Voltammetry (including anodic and cathodic stripping) or DC, Differential and Intermittent Pulse Amperometry); incorporate broad operational flexibility via RS232 interface and computer software; and are capable of stand-alone operation without computer.

However, the invention is not so limited and other monitors may be used, such as potentiometers and electrochemical analyses. Thus, the electronics 200 in Figure 2 of the amperometric monitor 112 shown in Figure 1 are, by way of example and illustration, but one means for imposing an electrical potential across the electrodes of the sensor 110 and but one means for detecting the resultant electrochemical signal.

The monitor 112, in the embodiment illustrated, includes a port 120 to accept the sensor 110, a button 122 to begin the analysis, and a liquid crystal display ("LCD") screen 124 to display results. The monitor 112 can display test results on its LCD screen 124 or store data in an internal memory not shown in Figure 1. The monitor 112 is configured with an RS-232 port 126 so that stored data can be later uploaded to a personal computer (not shown) or network (also not shown) for further analysis or long-term storage should that be desired.

Figure 2 is a block diagram of the monitor 112's electronics 200. Wiring diagrams for this particular embodiment are set forth in United States Letters Patent 5,873,990, entitled "Handheld Electromonitor Device,"issued February 23,1999, to Andcare, Inc. as assignee of the inventors Wojciechowski, et al. For clarity, some selected portions of that patent are reproduced below.

Returning to Figure 2, the electronics 200, in this particular embodiment, are built around a MOTOROLA MC6805 main processor 210. Other processors may be used in alternative embodiments. The MC6805 is an 8-bit microprocessor with 176 bytes of internal random access memory ("RAM"), 8Kb of program memory space, 24 I/O lines, 2 serial

interfaces, and a hardware timer. The 24 I/O lines and one serial port are used to connect to the external components. The second serial port allows a host computer to communicate with the system using a standard interface such as the RS-232 interface 126.

The electronics 200 include a digital-to-analog ("D/A") converter 212 to generate a known voltage and an analog-to-digital ("A/D") converter 214 to measure the current.

Additional operational amplifiers generate the counter electrode voltage and measure the reference voltage of the sensor and convert the current to a voltage for the A/D. An analog switch (not shown) disconnects the electronics from the sensor connector 218 when no sensor is installed. The data collected while processing the sample is stored in the 8Kb random access memory ("RAM") 220 for later analysis. An electrically erasable, programmable, read-only memory ("EEPROM") 222 may be used to store the parameters for the measurement process.

A lookup table, if incorporated into the device, translates the result to the final displayed value in the LCD 124. A power supply 226 for the system comprises either internal batteries or a 120V AC power module. The commercial 16-character LCD 124 may be used to display messages and the final result of the measurement.

A software support program for the monitor 112 allows a user to set the different parameters associated with the process. These parameters include, in the present invention, timing and voltage levels for each state and frequency of square wave modulation used in the scan state. In addition, the data may be uploaded from the instrument and displayed. The firmware can be divided into measurement of the data and communications to a host computer.

Used in the context of the present invention, and as generally understood by those skilled in the art, firmware refers to the software used as part of the disclosed device; that is, the software that is firmly fixed in the apparatus and which has been especially developed for the embodiments disclosed and described herein. The software is discussed more fully below in connection with the operation of the monitor.

This monitor 112 is sufficiently compact to be hand-held and operates on samples mounted on the printed circuit board, biosensor 110. The sensor 110 is inserted through a small opening 114 into a box-like structure 118. The sensor 110 holds a sample, i. e., a target nucleic acid segment/probe hybrid. This particular monitor 112 imposes an electrical potential across at least two electrodes on the sensor. The imposed potential will generate an electrical current if the target is present. This process is known as"amperometry,"and is discussed more fully

below. From the generated current, the presence of the target can be detected. In some embodiments, generated currents can be used not only to detect the presence of a target, but also its identity and quantitation. The table immediately below sets forth a variety of structural and operation characteristics for the particular embodiment of the monitor 112 illustrated in Figure 1. Size10.2 cm W x 5. 7 cm H x 19. 3 cm D in. H x 7.7in.D) Weight(withoutbattery)347g Operatingtemperaturerange12°Cto40°C(54°to104°F) Powerrequirementsstandard9VBattery(6.8Vminimum) orACAdapter(9-12VDC,100mAminimum) Sensorconnectorconfigurationleftcontactreferenceelectrode centercontact working electrode right contact counter electrode Sensor/electrodecompatibilityANDCARESensorsoranyelectrodeswi th suitableconnectorinterface ComputercompatibilityIBM-typePCwithRS232port OperatingmodesDCAmperometry(DCA DifferentialPulseAmperometry IntermittentPulseAmperometry Delaytime0to600 s Delay potential-2000to+2000mV(ordisconnect) Precondition#1time0to600s Precondition#1potential-2000to+2000mV Precondition#2time0to600s Precondition#2potential-2000to+2000mV Precondition#3time0to600s Precondition#3potential-2000to+2000mV Measurementpotential(DCAmp.mode)-2000to+2000mV Basepotential(PulseAmp.mode)-2000to+2000mV Pulsepotential (PulseAmp.mode)-2000to+2000mV Totalmeasurementtime1to999s Currentsamplingfrequency1to25Hz Pulsetime(PulseAmp.mode)2to(1000/frequency)ms Ramp0.01to2.5V/s OCP(opencircuitpotential)-2000to+2000mV Maximumoutputcurrent5mA Currentrangesandcorrespondingformatsof1uAX.XXXuA LCDdisplayofcurrent10 I1A _XX.XX11A 100 pA +XXX. X I1A 1mAX.XXXmA Baselinenoise<0.1%(asRSDoffullcurrentrange) Size10.2 cm W x 5. 7 cm H x 19. 3 cm D (4. 0in. Wx2. 3in. Hx7. 7in.D) Weight(withoutbattery)347g Operatingtemperaturerange12°Cto40°C(54°to104°F) Powerrequirementsstandard9VBattery(6.8Vminimum) orACAdapter(9-12VDC,100 mAminimum) Sensorconnectorconfigurationleftcontact reference electrode centercontact working electrode right contact counter electrode Sensor/electrodecompatibilityANDCARESensorsoranyelectrodeswi th suitableconnectorinterface Delay time0to600 s Delaypotential-2000to+2000mV(ordisconnect) Precondition#1time0to600s Precondition#1potential-2000to+2000mV Precondition#2time0to600s Precondition#2potential-2000to+2000mV Precondition#3time0to600s Precondition#3potential-2000to+2000mV Measurementpotential(DCAmp.mode)-2000to+2000mV Basepotential(PulseAmp.mode)-2000to+2000mV Pulse potential (PulseAmp.mode)-2000to+2000mV Totalmeasurementtime1to999s Currentsamplingfrequency1to25Hz Pulsetime(PulseAmp.mode)2to(1000/frequency)ms Ramp0.01to2.5V/s OCP(opencircuitpotential)-2000to+2000mV Maximumoutputcurrent5mA Currentrangesandcorrespondingformatsof1pAX.XXXpA LCDdisplayofcurrent10uAXX.XXpA 100 pA _XXX. X pA 1mAX.XXXmA Baselinenoise<0.1%(asRSDoffullcurrentrange)

The Sensor. The structure of the printed circuit board, biosensor 110 is illustrated more clearly in Figure 3. More particularly, Figure 3 depicts one specific embodiment of the sensor 110 including a reference electrode 305 and a working electrode 310 printed on a printed circuit board ("PCB") substrate 315. The sensor 110 also includes a sample well 325, where the hybridized sample is placed for analysis.

The electrodes 305,310 are printed on the PCB substrate 315 by depositing a conductive material, preferably a bulk or colloidal metal, on the PCB substrate 315. Electroplating techniques known to the art of PCB fabrication may be used. Exemplary bulk or colloidal metals include gold and silver, but the invention is not so limited. The working electrode 310 defines the sample well 325 in some embodiments. The sample well 325 may also be created

by etching away material from the PCB substrate 315 using etching techniques known to the art of PCB fabrication. In some embodiments, both of these techniques may be used to define the sample well 325. Because the electroplating and etching techniques are known to the PCB fabrication art, those in the art of the present invention may readily obtain embodiments of the sensor 110 from PCB fabricators given the disclosure herein.

The present invention admits significant variation in the construction of the reference and working electrodes 305,310. Colloidal metals, such as gold, provide an excellent surface for electromagnetically binding the hybridized nucleic acid segments to the working electrode 310. Bulk metals, however, provide surfaces markedly less suitable for this binding. If the working electrode 310 comprises an electroplated bulk metal, it should be coated with a material more conducive to the present process, i. e., one that facilitates the electromagnetic binding of the hybridized molecules to the working electrode. Such a nucleic acid, diffusable coating may include carbon, for instance. The reference and working electrodes 305,310 may be constructed from different materials, e. g., a silver reference electrode 305 and a gold working electrode 310.

One significant variation on the embodiment in Figure 3 includes conjugating with the metal a material facilitating the electrochemical bonding of the hybridized nucleic acid segment to the working electrode 310. One such material is streptavidin, an avidin derivative. Other suitable materials, including other avidin derivatives, may also be employed.

Another significant variation on the particular embodiment in Figure 3 includes a third, or counter, electrode. Figures 4A-4F illustrate several embodiments including this variation.

The counter electrodes 410 permit quantitation of the hybridized nucleic acid segments. The counter electrodes 410 may be constructed in the same manner using the same materials as discussed above for the reference and working electrodes 305, 310.

Yet another significant variation is specifically illustrated in Figure 4F. This sensor 1 lOf includes an array of sample wells 325 and associated electrodes 305,310, and 410. Using such an array, one may analyze a plurality of samples from the same source to test for a variety of nucleic acid segments. One may also analyze a plurality of samples from different sources to test each sample for the same nucleic acid segment. The particular embodiment of the monitor

112 in Figure 1 is not capable of use with an arrayed sensor such as the sensor 1 lOf. However, embodiments of the monitor 112 that can are also commercially available from the same source.

Using the PCB substrate 315 provides a number of advantages over the state of the art not readily apparent to those skilled in the art. For instance, screen printing can achieve only inferior resolution relative to PCB fabrication, which becomes an issue when implementing embodiments, such as the sensor 110f in Figure 4F, employing arrays of sample wells and electrodes. This unconventional use of PCB substrates had not previously been employed in the present art, largely because of the conventional wisdom against these types of sensors.

However, subsequent investigation showed that PCB substrates are sufficiently inexpensive, that they are more rugged and less susceptible to damage, and that PCB manufacturing techniques provide resolutions superior to those of screen-printing.

In certain cases, the working electrode 310 bound to an immobilized capture probe/target-nucleic acid segment a detector probe which is conjugated to an electroactive reporter group, for example horseradish peroxidase ("HRP"), which can transfer electrons.

When suitably combined with a reference and a working electrode 305,310, the capture probe/target-nucleic acid segment/detector probe/electroactive reporter group complex, in the presence of a substrate or activator for the electroactive reporter group, such as peroxide, and an electron mediator, causes a measurable and quantifiable electrical current.

The Probes. The electrochemical detection system 100 of the present invention employs biological probes. These probes may also be described as gene-probes, nucleic acid- probes, DNA-probes, oligonucleotide-probes, or any of several other labels. Generically, they are referred to in this description as simply"probes."The present invention typically comprises a first probe, the"capture probe."Some embodiments may also comprise a second probe, the "detector probe. "Optionally, three, four, five or more probes may be used in still other embodiments. However, in some embodiments, it may be desirable to use only a single biological probe.

As used herein, each probe comprises at least an oligonucleotide segment, which is complementary to a contiguous nucleic acid segment of an identified, target pathogen such that the oligonucleotide segment specifically hybridizes to the nucleic acid segment of the pathogen under conditions of high stringency. Oligonucleotide segments of 18 to 50 nucleotides are

preferred; however, shorter or longer segments may in certain instances be employed ; e. g., 15, 16,17, etc. or even 51,52, etc. as well as any number of 19,20,21, etc. up to 50 or so nucleotides.

As used herein, the term"complement"refers to the strand of nucleic acid that will hybridize to a first nucleic acid segment to form a double stranded molecule under stringent conditions. Stringent conditions are those that allow hybridization between two nucleic acid segments with a high degree of homology, but preclude hybridization of random segments. For example, hybridization at low temperature and/or high ionic strength is termed low stringency and hybridization at high temperature and/or low ionic strength is termed high stringency. The temperature and ionic strength of a desired stringency are understood to be applicable to particular probe lengths, to the length and base content of the segments and to the presence of other compounds such as formamide in the hybridization mixture.

Thus, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target segment. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e. g., conditions of high stringency where one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCI at temperatures of about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature.

Thus, hybridization conditions can be readily manipulated, and conditions can be readily selected depending on the desired results.

Short stretches (about 18-50 nucleotides) of single stranded DNA are preferred as oligonucleotide components of the probes of the invention. Two oligonucleotides directed toward separate, non-overlapping segments or regions of a target nucleic acid segment are used in a sandwich hybridization format. Using two non-overlapping probes to identify a target microorganism reduces the risk of"background noise"being interpreted as a false positive reading, as compared to a system that relies on the hybridization of a single probe for detection.

In certain embodiments, the oligonucleotide segment of a probe, either capture or detector, may be bound, bonded, conjugated or otherwise coupled with either a protein, such as biotin, or an antibody; or with another molecule, such as fluorescein ("FL") or dioxigenen ("DIG"), that is able to bond with an electroactive reporter group or an electroactive label, such as horseradish peroxidase ("HRP"). Atlernatively, the probe may be bonded directly to a sensor 110's electrode 310. Alternatively, the probe may be able to hybridize to another molecule, such as an oligonucleotide or protein (e. g., an avidin or avidin derivative, such as streptavidan, or a protein, such as protein G or protein A), which is bound to the sensor 110's electrode 310. Permutations of these alternatives may also be used in some embodiments.

Thus, while embodiments of the invention employ only a capture probe, more typical embodiments will include a capture probe and a detector probe. In some of these embodiments employing both a capture probe and a detector probe, the capture probe may be biotinylated and is readily bound to the colloidal gold-streptavidin conjugate of the working electrode array. The detector probe is labeled with fluorescein. A preferred detector probe comprises at least one molecule of fluorescein, a more preferred detector probe includes about two to about eight molecules of fluorescein and a particularly preferred detector probe includes about two to about four molecules of fluorescein. Anti-fluorescein antibody conjugated to an electroactive label, e. g., horseradish peroxidase ("HRP"), may be used to enzyme-label the detector probe in certain instances.

A preferred source of target DNA is ribosomal DNA ("rDNA") rather than genomic DNA ("gDNA") because of its far greater copy number in a given cell. For example, there are approximately 1000 copies of a rDNA gene per E. coli cell and approximately 500 copies of an rDNA gene per eukaryotic cell. However, the source of target nucleic acid segment may alternatively be gDNA, ribonucleic acid ("RNA"), chromosomal DNA ("cDNA"), messenger RNA ("mRNA") or ribosomal RNA ('rRNA").

It is preferred that double stranded DNA be denatured before hybridization. DNA may also be treated with restriction enzymes before hybridization with the probes. The stability of the hybrids and consequently the specificity of hybrid formation can be adjusted by varying the temperature and ionic strength of the solution. Other conditions that may be desirable to optimize include, but are not limited to, temperature, magnesium concentration, amount of target DNA and probes, and interference between hybridization and sensitivity.

In general, treatment with a standard lysis buffer, preferably 0.75N sodium hydroxide, is all that is required to lyse bacteria, in cultivable or uncultivable states, and release the target- DNA in a form that may be detected with the claimed invention. Optimal lytic conditions may require appropriate adjustment of pH, treatment time, and possibly additional lysis and sample modifying reagents. As is common for analysis, additional modifiers may be desired to improve the performance of the system. For example, the addition of AQ polymer improves the spreading of water samples over the surface of the test electrode, thereby improving the sensor performance.

In certain embodiments where at least two probes are used, either the capture probe or the detector probe may hybridize to any one of a generic group of related targets of interest. In such cases, it is generally preferred that the other probe be specific to a particular target species.

Alternatively, one may choose to use both a capture probe and a detector probe that hybridize to any one of a group of related targets if the objective is to measure the occurrence of a particular generic group. For example, a detector probe can hybridize to 16s rDNA of both E. coli and Salmonella, and a first capture probe can also hybridize to 16s rDNA of both E. coli and Salmonella. An electrochemical signal generated by using these two probes would indicate that either or both E. coli and Salmonella are present in the sample. Alternatively, a second capture probe which only hybridizes to nucleic acid segments of E coli could be used in place of the first capture probe. An electrochemical signal generated by using the detector probe and the second capture probe would only indicate the presence of E. coli in the sample. Salmonella nucleic acid segments that are hybridized to the detector probe would not be detected, since these the Salmonella nucleic acid segments are not coupled to the electrode.

Operation of the Apparatus As noted above, the present invention also provides an electrochemical detection system that uses only a capture probe. In this instance, the electrochemical signal is generated when the target-probe hybrid is immobilized on the working electrode. An amperometric potential is applied across the reference and working electrodes. An electrochemical current flows between the working electrode and another electrode in the presence of an electroactive group that is either inherent in the target nucleic acid segment, e. g., a thiol group, or is chemically added to the target. The discussion below of the operation of the apparatus 100 in Figure 1, however, pertains to an embodiment in which a three-electrode sensor is used with two probes. This

embodiment is used for clarity in teaching the use and application of a more typical embodiment of the the invention. However, the invention is not so limited.

The measurement of data from the sensor 110 is based on connecting the sample to a voltage source housed in the monitor 112 as described further below and measuring the current.

The firmware flowchart in Figure 5 illustrates the steps in the process. Each step has an associated time duration set by the user. This time can be set to zero, skipping that part of the process. In addition, each step has a voltage applied to the sensor during that time. The flowchart shows that the routine starts in a loop, waiting for the"START"switch 122, in Figure 1, to be pressed. Once the switch 122 is activated, each step is sequential. If the time is set to zero, that step is skipped. The present system supports four stages: Initial delay (box 510), Precondition #1 (box 520), Precondition #2 (box 530), and Deposition (box 540).

The next step is called the Scan stage (box 550). This stage is more complex. The voltage is applied depending on the amperometry technique used, as is discussed more fully below. When the scan stage is completed, the data analysis routine (box 560) calculates the difference between these two currents and using this data, calculates the analyte level. Finally, the data is displayed (box 570) on the LCD display 124.

In one embodiment, electrochemical detection may be achieved using conventional DC Amperometric detection. Most electrochemical systems employing electrochemical sensors such as those for blood glucose, oxygen, or hydrogen peroxide, as well as immunosensors and gene-probe sensors, measure currents using DC Amperometric Detection, which is also referred to as"Amperometric Detection."In this method, a constant electrical potential is continuously applied across two electrodes and, after allowing sufficient time (usually, several seconds), a "steady state"current is measured. The current results from an electrochemical process induced by the applied potential. In this induced electrochemical process, the analyte electrochemically either reacts at the working electrode or is involved in a reaction cycle with some other species reacting electrochemically. In order to have analytical utility, the measured current signal has to have predictable and stable correlation to the concentration of the analyte.

However, the major drawback of DC Amperometric Detection is that the properties of the electrode interface change in time as a result of continuously applied potential. The change, usually called electrode fouling, may be due to adsorption of sample components (e. g., proteins,

lipids) on the surface of the sensor which changes the current signal and makes it less predictable. The measurement tends to be slow as sufficient time must be allowed for the electrode surface to equilibrate with the tested solution.

Thus, in one aspect of the invention, an innovative form of Amperometric Detections known as Pulsed Electrochemical Detection ("PED") is used. PED is currently used almost exclusively in the unrelated art of high performance liquid chromatography ("HPLC"). PED is an amperometric detection method in which a potential waveform such as the signal 600 in Figure 6 is imposed on the working electrode in a detector system. It is a non-stationary system. That is, solution containing analytes flows through a cell and passes by the detector.

Despite its popularity in HPLC, PED, or for that matter any other kind of Pulse Amperometric Detection, has not been used with stationary systems such as those involving electrochemical sensors.

In PED, the potential is applied as a series of fast (i. e., about 50-400 ms) pulses of constant amplitude. The current is measured at the end of the detection pulse while other pulses, of similar or higher amplitude, may be used to precondition the electrode surface either by electrochemical cleaning (i. e., desorption) or reactivating (i. e., regeneration of active groups on the electrode surface). The advantage of this approach is that the electrode fouling due to adsorption of the product of electrochemical reaction, or other sample components, can be greatly diminished.

In this aspect of the present invention, two new PED approaches are employed in alternative embodiments. These two new approaches are denominated Differential Pulse Detection ("DPD") and Intermittent Pulse Detection ("IPD"). These two approaches have been developed because the limitations of the preceding detection systems did not allow detection of pathogens either with sufficient sensitivity to eliminate culturing steps or in a stationary system.

Differential Pulse Detection ("DPD") applies a series of two pulses as shown by signal 605 in Figure 6. The first pulse is a longer"base"pulse set at the"resting"potential, ie., where no significant charge transfer should be expected, or at potential that would allow measurement of a background current. The second pulse is a shorter detection pulse with sufficient potential to electrochemically oxidize or reduce the analyte, or one of the products or reagents participating in the analyte reaction in solution phase or on the electrode surface. The resulting

current is measured at the end of both pulses and the subtracted value is used as a signal. By proper selection of the base potential, variable effects of background currents are eliminated.

Intermittent Pulse Detection ("IPD") differs significantly from DPD in that, instead of the"base"pulse during which controlled potential is applied, the working electrode is disconnected from the potentiostatic circuit of the monitor 112. An exemplary signal for such an applied voltage is the signal 610 in shown in Figure 6. Thus, the electrode is allowed to "relax"or"rest,"i. e., assume its natural potential where truly no charge transfer is occurring.

The boundary conditions are restored in a natural, i. e., not imposed by any applied potential, way. The current measured at the end of the detection pulse therefore reflects the activity of the enzyme (or other redox) label attached to the electrode surface, and is not obscured by the accidental charge transfer process. Such accidental charge transfer could occur, however, when the base potential selected in the DPD is not truly the open circuit potential. IPD greatly improves the sensitivity of the present invention. This is surprising in that one would not expect that disconnecting the electrode would actually improve its function and the sensitivity of the assay.

IPD measurements involve a series of millisecond double-step pulses comprising of a base potential pulse followed by a detection potential pulse without interrupting the contact with the potentiostat between the pulses. Base pulses have a distinctively different potential relative to the detection pulse. Base pulsing can be used to precondition the surface before each detection pulse or to restore the boundary conditions disturbed by the processes taking place during detection pulse. For example, +200 mV potential can be used as base potential and-100 mV. Consecutive double-step pulses are separated by a longer period when the electrode is disconnected from the potentiostat circuit.

Currents are measured during last 100 microseconds of the base pulse and the detection pulse. Current measured at detection pulse, or the difference between the currents measured at detection pulse and base pulse, can be used as current signal for the purpose of detecting the presence or determining the quantity of the target in test sample. The effect of intermittent double pulse is an increase in sensitivity of measurement and additional reduction of electrode fouling effect due to the interferants or surface reaction products adsorbing or otherwise distorting the performance of sensor.

Advantages of both differential and intermittent pulse measurement schemes include: électrode fouling is effectively eliminated. The measurement time, when a potential is applied to the electrode, is significantly reduced (from seconds to milliseconds) and, consequently, adsorption of reaction product (s) or other sample components is minimized, helping to maintain a steady response of the electrode.

The measurement is faster. Currents are measured on the millisecond time scale while the time scale of seconds or minutes is used in conventional DC Amperometric Detection. One to two seconds is sufficient to establish a"steady state"current signal and a quantifiable measurement.

A high rate of current measurement (about 5-50 Hz can be used, but about 10 to 25 Hz sampling rates are preferred) allows for rapid acquisition of a large numbers of data points and effectively reduced background signal interference.

Additional improvement of the signal-to-noise ratio can be accomplished by data averaging or fast Fourier transformation ("FFT") smoothing.

Figures 7A-7B illustrate the operation of the probe and sensor of the present invention in one particular embodiment. This particular embodiment employs a three-electrode sensor and a detector probe conjugated to an electroactive reporter group. However, the invention is not so limited, since not all embodiments employ a capture probe or an electroactive reporter group as is discussed more fully above. More particularly, Figures 7A-7B schematically diagram the configuration of probes and labels for the capture and detection of nucleic acid segments on the surface of electrochemical sensors and a series of oxidation/reduction reactions initiated by horseradish peroxidase ("HRP"). The series of reactions generates a mediated catalytic current on the surface of the sensor (center of the figure) for a three-electrode-sensor format. As noted above, not all embodiments employ a capture probe Kits.

The invention also provides easy-to-use kits which contain monitors, reagents and procedures that can be utilized in a clinical or research setting or adapted for either the field laboratory or on-site use. These kits can be widely employed in less technologically developed areas or countries which do not have well-equipped laboratories and at remote sites far from well-equipped laboratory facilities. The invention thus is useful in monitoring for the presence

of microbes which cause waterborne diseases at both water treatment plants and at untreated water supplies such as a river or lake.

A Method of Using the Apparatus The present invention provides a method of using the system 100 for the quantitative detection of nucleic acid segments in a sample. The source of the sample may be food, water or a biological or clinical sample which can be treated such that (1) material suspected of containing nucleic acid segments from the microbes can be freed into an aqueous medium and subjected to lytic conditions designed to release the nucleic acid segments characteristic of the microbes from the material into the aqueous medium, and (2) the capture and detector probes hybridize with their respective characteristic target nucleic acid segments. The aqueous medium is such that an electrochemical signal is generated by the hybridized probes/nucleic acid segments when an electrical potential is imposed thereacross. The resulting electrochemical signal is detected by a printed circuit board, biosensor. In certain cases, an electron mediator and an electroactive reporter group are used to help generate an electrochemical signal.

The general electrochemical detection method for this particular embodiment is described by the following equations, together with Figures 7A and 7B: probe + nucleic acid target specific hybrid specific hybrid + enzyme conjugate enzyme-labeled species.

The enzyme-labeled species is captured at the sensor surface, as shown schematically Figures 7A, and a current is generated upon addition of enzyme substrate, as shown schematically in Figure 7B.

The human interaction with the monitor and sensor of the apparatus is illustrated in Figures 8A-8C. The assay chemistry can be conveniently divided into four general steps: (1) sample treatment/cell lysis, (2) hybridization, (3) hybrid capture, and (4) detection. The sample is first treated. The sensor is then removed from the monitor 112, Figure 8A, and the sample deposited in a well of the sensor 110, Figure 8B. During this period, hybridization and hybrid detection occur. The sensor 110 is then placed in the slot of the monitor 112, Figure 8C, whereupon the detection occurs. Each of the assay chemistry's four general steps is discussed more fully below.

Sample Treatment/Cell Lysis Samples are collected and concentrated or lysed, as required. Sample concentration is accomplished by a membrane filter technique accepted by the United States Environmental Protection Agency for microbiological testing of potable water (11th edition, Standard Methods for the Examination of Water and Wastewater). In traditional assays the filter is placed in a petri dish with nutrient media for several days for growth of bacterial colonies. Such culturing steps are not typically required by the present invention. Appropriate adjustment of pH, treatment time, lytic conditions and sample modifying reagents may be altered in order to optimize reaction conditions. Such modification techniques are well known to those of skill in the art. Although culturing steps are generally undesirable, procedures that include short term culturing prior to measurement may be desirable in some cases. Such assays by the disclosed method would still have shortened analysis time compared to conventional assays, because it is unnecessary to grow organisms to the point of visible colonies.

Hybridization Hybridization of the target DNA and oligonucleotide probes is generally carried out in an aqueous solution which contains an excess amount of probe, or alternatively contains a detector probe if the capture probe is immobilized to the working electrode of the sensor and two probes are used. In either case, hybridization proceeds rapidly because both the target DNA and probes are in a homogeneous hybridization system rather than a heterogeneous hybridization system. In a homogeneous hybridization system, hybridization of probes and target DNA occurs in solution and the resultant hybrid does not need to be transferred to a solid support surface, such as a nitrocellulose filter, in order for detection to take place.

In conventional filter hybridization methods, such as Southern blot hybridization, the target DNA or RNA is immobilized on a filter membrane and then allowed to react with the probe molecule. After the conventional hybridization reaction is complete, excess probe molecules are removed by washing the filter and the labeled hybrids are detected by autoradiography or by a non-radioactive detection method. In these conventional methods the rate of hybridization is very slow because the target is present in low concentration and immobilized on a surface. Overnight or longer incubations and autoradiographic exposures are sometimes required to obtain good sensitivity. Protocols of the claimed invention are faster, because they are designed to increase hybridization rates by carrying out reactions in solution

and capturing the DNA hybrids afterwards. Thus the amount of time, labor, expense and technical expertise required is reduced.

The process of selecting and preparing nucleic acid segments either as capture or detector probes in nucleic acid biosensor methods is well-known to those in the molecular biological arts. Such nucleic acids (often called"probes"or"primers"or"oligos"etc.) may be prepared e. g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCRTM technology of U. S. Patent 4,683,202, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the nucleic acid probes of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the target gene, gene fragment, or polynucleotide to be detected. Depending on the application envisioned, one may employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e. g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating target polynucleotides.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate one or more selected sequences, functional equivalents, or the like, less stringent (reduced stringency) hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ salt conditions such as those of from about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally

appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

SYNTHESIS AND DETECTION OF RIBOZYMES In one embodiment, the biosensors disclosed herein may be utilized to detect specific target nucleic acids such as ribozymes in a sample. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity as is known in the art. See, e. g., Kim and Cech, "Three-dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena,"Proc. Natl. Acad. Sci. U S A, 84 (24): 8788-8792,1987; Gerlach et al., "Construction of a plant disease resistance gene from the satellite RNA of tobacco rinspot virus,"Nature (London), 328: 802-805,1987; Forster and Symons,"Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites,"Cell, 49: 211-220,1987.

For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. See Cech et al.,"In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence,"Cell, 27 (3Pt2): 487-496,1981; Michel and Westhof,"Modeling of the three- dimensional architecture of group I catalytic introns based on comparative sequence analysis," J. Mol. Biol., 216: 585-610,1990; Reinhold-Hurek and Shub,"Self-splicing introns in tRNA genes of widely divergent bacteria,"Nature, 357: 173-176,1992. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then

binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.

The enzymatic nucleic acid molecule may be formed in a hammerhead, hairpin, a hepatitis virus, group Iintron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al.

(1992). Examples of hairpin motifs are described by Eur. Pat. Appl. Publ. No. EP 0360257); Hampel and Tritz, Biochem., 28: 4929,1989; Hampel etal., Nucl. Acids Res., 18: 299,1990; and U. S. Patent 5,631,359. An example of the hepatitis virus motif is described byPerrotta and Been, Biochem., 31: 16,1992; an example of the RNaseP motif is described by Guerrier- Takada et al., Cell, 35: 849,1983; Neurospora VS RNA ribozyme motif is described by Saville and Collins, Cell, 61: 685-696,1990; Saville and Collins, Proc. Natl. Acad. Sci. U S A, 88: 8826-8830,1991; Collins and Olive, Biochem., 32: 2795-2799,1993; and an example of the Group I intron is described in U. S. Patent 4,987,071.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, and synthesized to be tested in vitro and in vivo, as described therein. Ribozymes may be designed to anneal to various sites in the mRNA message, and can be chemically synthesized. The method of synthesis used follows the procedure for normal RNA synthesis as described in Usman etal., J. Am. Chem. Soc., 109: 7845-7854,1987, and in Scaringe et al., Nucl. Acids Res., 18: 5433-5441,1990, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5- end, and phosphoramidites at the 3-end. Hairpinribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992). Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2-amino, 2-C-allyl, 2-flouro, 2-o-methyl, 2-H (for a review see e. sman and Cedergren, 1992).

SYNTHESIS AND DETECTION OF PROTEIN NUCLEIC ACIDS In certain embodiments, it may be desirable to utilize the biosensors of the present invention in the detection of peptide nucleic acids (PNAs), or alternatively, use PNAs as either as an oligonucleotide for the capture probe and/or the detector probe (s) in the detection of target polynucleotides. The synthesis, design, and use of PNAs in hybridization assays are well- known to those of skill in the molecular biological arts. A PNA is a DNA mimic in which the

nucleobases are attached to a pseudopeptide backbone. See, e. g., Good and Nielsen, Antisense Nucleic Acid Drug Dev., 7 (4): 431-437,1997.

PNAs may be utilized in a number of methods that have traditionally employed RNAs and/or DNAs. Often PNA sequences perform better in techniques than the corresponding RNA or DNA sequences and have utilities that are not inherent to RNA or DNA. PNAs may be utilized with the disclosed biosensors either as capture probes or detector probes when target nucleic acids are present in the sample, or alternatively, PNAs themselves may be the target nucleic acids sought to be identified in a given sample.

PNAs have 2-aminoethyl-glycine linkages replacing the normal phosphodiester backbone of DNA. See, e. g., Nielsen, Egholm, Berg, Buchardt, Science, 254: 1497-1500,1991; Hanvey et al., Science, 258: 1481-1485,1992; Hyrup and Nielsen, Bioorg. Med. Chem., 1996; Neilsen, In: Perspectives in Drug Discovery and Design 4, Escom Science Publishers, pp. 76- 84,1996. This chemistry has three important consequences: firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAs are neutral molecules; secondly, PNAs are achiral, which avoids the need to develop a stereoselective synthesis; and thirdly, PNA synthesis uses standard Boc, see Dueholm et al., J. Org. Chem., 59: 5767-5773,1994, or Fmoc, see Thomson et al., Tetrahedron, 51: 6179-6194,1995, protocols for solid-phase peptide synthesis, although other methods, including a modified Merrifield method, have been used, see, e. g., Christensen et al., J. Pept. Sci., 1 (3): 175-183,1995.

PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems (Framingham, MA, USA). PNA syntheses by either Boc or Fmoc protocols are straightforward using manual or automated protocols, see, e. g., Norton, Waggenspack, Varnum, Corey, Bioorg. Med. Chem., 3: 437-445,1995. In contrast to DNA and RNA, each of which contains negatively charged linkages, the PNA backbone is neutral. In spite of this dramatic alteration, PNAs recognize complementary DNA and RNA by Watson-Crick pairing, see, Egholm et al., Nature, 365: 566-568,1993, validating the initial modeling by Nielsen, Egholm, Berg, Buchardt, Science, 254: 1497-1500,1991. PNAs lack 3'to 5'polarity and can bind in either parallel or antiparallel fashion, with the antiparallel mode being preferred, Egholm et al., Nature, 365: 566-568,1993.

Hybridization of DNA oligonucleotides to DNA and RNA is destablized by electrostatic repulsion between the negatively charged phosphate backbones of the complementary strands.

By contrast, the absence of charge repulsion in PNA-DNA or PNA-RNA duplexes increases the melting temperature (Tm) and reduces the dependence of Tm on the concentration of mono-or divalent cations, Nielsen, Egholm, Berg, Buchardt, Science, 254: 1497-1500,1991. The enhanced rate and affinity of hybridization are significant because they are responsible for the surprising ability of PNAs to perform strand invasion of complementary sequences within relaxed double-stranded DNA.

PNAs have many uses; methods of characterizing the antisense binding properties of PNAs are discussed in Rose, Anal. Chem., 65 (24): 3545-3549,1993, and Jensen et al., Biochemistry, 36 (16): 5072-5077,1997. Rose uses capillary gel electrophoresis to determine binding of PNAs to their complementary oligonucleotide, measuring the relative binding kinetics and stoichiometry. Similar types of measurements were made by Jensen et al. using BIAcoreTM technology. Other applications of PNAs include use in DNA strand invasion, Nielsen, Egholm, Berg, Buchardt, Science, 254: 1497-1500,1991; antisense inhibition, Hanvey et al., Science, 258: 1481-1485,1992; mutational analysis, Orum, Nielsen, Egholm, Berg, Buchardt, Stanley, Nucl. Acids Res., 21: 5332-5336,1993; enhancers of transcription, Mollegaard, Buchardt, Egholm, Nielsen, Proc. Natl. Acad. Sci. USA, 91: 3892-3895,1994; nucleic acid purification, Orum, Nielsen, Jorgensen, Larsson, Stanley, Koch, BioTechniques, 19: 472-480,1995; isolation of transcriptionally active genes, Boffa, Carpaneto, Allfrey, Proc.

Natl. Acad. Sci. USA, 92: 1901-1905,1995; blocking of transcription factor binding, Vickers, Griffith, Ramasamy, Risen, Freier, Nucl. Acids Res., 23: 3003-3008,1995; genome cleavage, Veselkov, Demidov, Nielsen, Frank-Kamenetskii, Nucl. Acids Res., 24: 2483-2487,1996; biosensors, Wang, J. Am. Chem. Soc., 118: 7667-7670,1996; in situ hybridization, Thisted, Just, Petersen, Hyldig-Nielsen, Godtfredsen, Cell Vision, 3: 358-363,1996; and in a alternative to Southern blotting, Perry-O'Keefe, Yao, Coull, Fuchs, Egholm, Proc. Natl. Acad. Sci. USA, 93: 14670-14675,1996.

SYNTHESIS AND DETECTION OF ANTIBODY COMPOSITIONS In certain embodiments, the biosensors of the present invention may be used in the detection and quantitation of target polypeptides in a sample. As such, the biosensor may be coupled with a capture molecule that specifically recognizes the target polypeptide to be detected. Exemplary capture molecules useful for the detection of peptides, peptide epitopes,

and/or polypeptides include protein affinity reagents and antibodies. In an illustrative embodiment, the biosensor may comprise an antibody operably linked to the electrode wherein the antibody comprises a detectable electrochemical label that produces a signal detectable by the electrode when the antibody specifically binds to its target polypeptide in a sample.

Means for preparing and characterizing antibodies are well known in the art. See, e. g., Harlow. and Lane, In: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988. In general, when an antibody-based biosensor is used for the immunodetection of a target polypeptide, the methods will generally include first obtaining a sample suspected of containing a target polypeptide, and contacting the sample with a biosensor that comprises an antibody specific for the target polypeptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). Once the primary immune complex is formed, an electrochemical signal is produced by the detector antibody (typically via the generation of an electrochemical signal that is detectable by the working electrode), and the presence of the target polypeptide in the sample is determined via the generation of a signal when an electrical potential is generated across the working and reference electrodes.

Contacting the chosen sample with the labeled antibody electrode under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply contacting the polypeptide sample with the biosensor as described herein. One may wish to incubate the mixture for a period of time sufficient to allow the detector antibody to form immune complexes with, i. e. to bind to, any target polypeptide present within the sample. After a specified time, the sample/electrode composition, may be washed with a suitable buffer or diluent to remove any non-specifically bound polypeptide species, allowing only those specifically bound species within the immune complexes to be detected.

Detection of the primary immune complexes using an antibody-based biosensor is based upon the detection of an electroactive label or alternatively with an enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase, glucose oxidase and the like also being suitable.

SYNTHESIS AND DETECTION OF POLYPEPTIDE COMPOSITIONS Means for preparing and characterizing polypeptides are also well known in the art. In general, when a polypeptide-based biosensor is used for the immunodetection of a target antibody, the methods will generally include first obtaining a sample suspected of containing a

target antibody, and contacting the sample with a biosensor that compriSesaoTypeptide having at least one epitope specific for the target antibody under conditions effective to allow the formation of an immunocomplex (primary immune complex). Once the primary immune complex is formed, an electrochemical signal is produced by the detector polypeptide (typically via the generation of an electrochemical signal that is detectable by the working electrode), and the presence of the target antibody in the sample is then detected via the generation of a signal when an electrical potential is generated across the working and reference electrodes.

Contacting the chosen sample with the labeled polypeptide or peptide-based electrode under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply contacting the sample suspected of containing the target antibody with the biosensor as described herein. One may wish to incubate the mixture for a period of time sufficient to allow the detector polypeptide to form immune complexes with, i. e. to bind to, any target antibody present within the sample. After a specified time, the sample/electrode composition, may be washed with a suitable buffer or diluent to remove any non-specifically bound antibody species, allowing only those specifically bound species within the immune complexes to be detected.

Detection of the primary immune complexes using a polypeptide-based biosensor is based upon the detection of an electroactive label or alternatively with an enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase, glucose oxidase and the like also being suitable.

The identification of particular polyepeptide epitopes and/or their functional equivalents is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U. S. Patent 4,554,101, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences. See, e. g., Jameson and Wolf,"The Antigenic Index: A Novel Algorithm for Predicting Antigenic Determinants,"Compu. Appl. Biosci., 4 (1): 181-6,1988; Wolf et al.,"An Integrated Family of Amino Acid Sequence Analysis Programs,"Compu. Appl. Biosci., 4 (1): 187-91,1988; U. S. Patent 4,554,101.

An epitopic core sequence is a relatively short stretch of amino acids that is "complementary"to, and therefore will bind, antigen binding sites on selected epitope-specific antibodies. An epitopic core sequence will elicit antibodies that are cross-reactive with antibodies directed against a particular target peptide. It will be understood that in the context of the present disclosure, the term"complementary"refers to amino acids or peptides that exhibit an attractive force towards each other.

The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U. S. Patent 4,554,101, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins see, e. g., Jameson and Wolf,"The Antigenic Index: A Novel Algorithm for Predicting Antigenic Determinants,"Compu. Appl. Biosci., 4 (1): 181-6,1988; Wolf et al.,"An Integrated Family of Amino Acid Sequence Analysis Programs,"Compu. Appl. Biosci., 4 (1): 187-91,1988 Computerized peptide sequence analysis programs (e. g., DNAStarTM software, DNAStar, Inc., Madison, WI) may also be useful in designing synthetic epitopes and epitope analogs in accordance with the present disclosure.

DIAGNOSTIC ASSAYS USING ELECTROCHEMICAL BIOSENSORS For diagnostic purposes, the nucleic acid-based, polypeptide-based, and antibody-based biosensors of the present invention may be utilized to detect and/or quantitate target biomolecules from a variety of samples. Indeed, it is envisioned that virtually any sample suspected of containing the selected target molecule of interest may be employed. Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed. This allows for the diagnosis of meningitis, otitis media, pneumonia, bacteremia and postpartum sepsis. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antibody or antigen samples, the detection of oligonucleotide compositions, the identification of cDNAs, reaction products, restriction fragments, single nucleotide polymorphisms, expressed sequence tags, nucleic acid vectors, or other delivery vehicles, or the selection of hybridomas, immunogenic sera, and the like.

Likewise, the clinical samples may be from veterinary sources and may include such domestic animals as horses, pigs, cattle, sheep, and goats. Samples from feline, canine, lupine, epine, and murine sources may also be used in accordance with the methods described herein.

Hybrid Capture The present invention allows identification of specific nucleic acid segments in crude samples. In certain cases, it is desirable to use two different probes, i. e., a capture and a detector probe. This hybridization technique can be more specific than single probe hybridization, because two hybridization events must occur in order to generate a signal. The capture probe electrodes are coated with molecules, such as streptavidin, avidin or even the capture probe, designed to capture target nucleic acid detector probe hybrids.

In embodiments which include a detector probe, a strong catalytic current is produced when target nucleic acid probe hybrids that are immobilized on the working electrode surface and labeled with an electrochemically detectable label, such as HRP, are exposed to an amperometric potential in the presence of a substrate, e. g., peroxide. The result of catalysis by the HRP label is a flow of current between the working electrode and another electrode, either the reference electrode in a two electrode system or the counter electrode in a three electrode system. The current is measured by a monitoring device, as referred to earlier.

Equations 1-3 illustrate the electrochemical detection of HRP bound at an electrode. An electron transfer mediator is used in this system. A preferred mediator is ferrocene monocarboxylic acid (Fc). electrode 2Fc++2e-2Fc(1) HOP.+2Fc o HRP,+2Fc+(2) HRPr+ H202, HRPo+2H20(3) At the molecular level, capture at the electrode of the sensor puts the HRP in close proximity to the working electrode, where it generates a current in the presence of peroxide. The peroxide is

added as a final step, equation 3, after allowing the probe and target segments to hybridize. The electrochemical detection assay exhibits rapid response with a good signal to noise ratio.

Another electron transfer mediator that can be used in the claimed invention is 3,3', 5,5'- tetramethylbenzidine.

The hybridization solutions can comprise buffers of relatively low stringency, as determined by previous hybridization studies.

In general, an analysis of the type disclosed above requires no more than about 10 or 15 minutes to perform. The signals generated are typically proportional to the amount (concentration) of target nucleic acid present in the sample.

Detection The sample containing hybridized target DNA/detector probe, or hybridized capture probe/target-DNA/detector probe or hybridized capture probe/target DNA, is applied to a sensor 110 as described above. A third, counter, electrode is also present if quantitation of microbial concentration is desired. Hybrids are captured at the working electrode surface by reaction of the capture probe with the electrode biosensor or by the hybridization of the target- DNA/detector probe hybrid with the capture probe that is already bound to the biosensor.

Although an excess of capture probe is present, either bound directly to the biosensor or in the aqueous hybridization solution, no catalytic current or electrochemical signal is generated by these non-hybridized capture probes because these capture probes are not also attached to an electroactive reporter. In one preferred embodiment, the capture probe is biotinylated and the electrode biosensor is coated with avidin, preferably a synthetic avidin.

The detection is actually performed by the monitor 112 operating as programmed on the hybridized sample held by the sensor 110. More particularly, the monitor 112 imposes an electrical potential across the electrodes of the sensor 110 and determines from the signal, or lack thereof, whether the sample contains the target nucleic acid segment. If desired, and if an appropriate embodiment of the sensor 110 is employed, then the target nucleic acid segment may be quantitated.

Distinct advantages of electrochemical detection combined with biological-probe methodology are readily apparent. The present invention shows sensitivity and selectivity equal to or better than conventional methods in a more rapid, less expensive and simpler-to-use format. A detectable signal can be generated in minutes as opposed to hours, as with many colorimetric assays, or even days, as with many radioassays. Harmful and increasingly difficult to dispose of materials, such as radioisotopes or mutagenic colorimetric labels, are not required.

Quantification of the signal is easily accomplished, and a pathogen has been successfully detected with as little as a picogram to femptogram (10-12 to 10-ils gram) of target DNA.

Coliform bacteria have been detected at ten-fold fewer pathogens than at the regulatory level of 200 cells per 100 ml of sample, i. e., Escherichia coli has been detected in a sample with as few as 200,000 E. coli cells/ml of aqueous sample.

Examples of pathogens that can be detected using the invention include, but are not limited to, bacteria such as Salmonella, Escherichia coli, Klebsiella pneumoniae, Bacillus, Shigella, Campylobacter, Helicobacter, Vibrio, parasites such as Giardia, Naegleria and Acanthamoeba and such as viruses Hepatitis and poliomyelitis.

By using specific segments of DNA or RNA that are characteristic of target microbes, pathogens can be unambiguously identified, regardless of their cultivable states, by direct analysis of contaminated food or water samples. Thus, more definitive data are provided regarding food and water quality, and the time-consuming culturing step associated with coliform counts is reduced or eliminated. In addition, distinctions can be made between different coliform bacteria, e. g., pathogenic v. nonpathogenic bacteria.

The invention is not only useful for the detection of food pathogens and water-borne microbes, but also may be employed to detect genetic variations associated with different disorders or diseases. Examples of diseases that can be detected by the present invention include cystic fibrosis, muscular dystrophy, sickle cell anemia, phenylketonuria, thalassemia, hemophilia, al-antitrypsin deficiency, disorders of lipoprotein metabolism and inherited forms of breast cancer. In addition, quantitative analysis of human genes is also desirable for analysis of amplified oncogenes and in the determination of gene expression levels in tumors.

The present invention improves upon current gene-probe assays by requiring fewer steps to perform, detecting specific targets at lower concentrations, and needing less time to

complete. A particular advantage of the present invention is that it can be used outside of a well-equipped laboratory setting. Complex instrumentation is not required because the probes and electrodes are employed with an inexpensive, hand-held meter or fieldable monitor. These electrochemical assays can be automated in a number of ways using relatively inexpensive equipment and procedures that are generally more robust and less complex for the operator to perform than comparable homogenous immunoassays.

The disclosed invention shows that probes may be effectively coupled to colloidal gold electrodes such that a target-DNA is detected with high sensitivity. Examples herein demonstrate the increased sensitivity achieved by the disclosed electrochemical probe methods as compared to conventional methods of detecting coliform bacteria in samples collected from marine/freshwater environments or food extracts. The electrochemical detection system of the present invention provides an improved means of monitoring human and environmental health through food and water-safety assays.

The quantifiable electrochemical signals of the invention result from electroactive groups being held in close proximity to a working electrode surface by an immobilized probe- target. A free electroactively labeled probe in solution that is not immobilized on the electrode surface does not couple efficiently enough to the electrode to produce an electrode response.

Thus, no separation of free probe from hybridized probe of the invention is needed.

The present invention significantly advances assay performance in terms of reduced time, complexity, and cost in an assay format that can be automated and multiplexed for high- throughput applications. Application areas include, but are not limited to, some of which are listed here: 1) kinetics of gene expression in different cell types under different stimulatory conditions; 2) comparison of pathogenic and non-pathogenic strains or of different organisms that share the same mechanisms of pathogenesis; 3) low-cost, high-throughput genetic tools for studies to relate gene expression of the pathogen with disease progression; and 4) studies of genotype and gene expression in non-infectious diseases, such as cancer.

The present invention meets the needs for increased sensitivity and throughput without requiring complicated techniques or large, expensive instruments. Furthermore, unlike current DNA chip assays, researchers using this"open"system can synthesize and use their own probes for the assays, rather than relying only on probe systems provided by the manufacturer. The

invention may find advanced applications in a wide range of biotechnology/biomedical laboratories and hospital clinics and in agricultural research institutions. It may become widely used in evaluation of gene expression, clinical diagnostics, DNA/RNA probe technology, DNA sequencing, and advanced DNA/RNA chips.

The versatility, as well as sensitivity and specificity, of this DNA detection system was studied in several research labs. The confirmatory assays involved detection of bacteria (e. g., E. coli and Salmonella), viruses (e. g., polio and hepatitis A), and single base mutations of medical importance (e. g., Factor V Leiden, BRCA1, Sickle Cell Anemia). The present invention can also be used to detect of Plasmodium falciparum.

With the monitor operating in the Intermittent Pulse Amperometric mode, nucleic acids can be detected with attomol sensitivity in 10 seconds or less. The present invention eliminates the need for gels, radioisotopes, and blotting. It is simple to use, inexpensive, and faster than conventional approaches. It can be conveniently operated in at least three variations: * a RAPID PCR-DETECT System is intended for detection and quantification of double-stranded PCR products directly, i. e., without a hybridization step; a a HYBRID PCR-DETECT System incorporates added specificity through selective hybridization of detector and/or capture probe; and * a DIRECT-DETECT System offers fast and sensitive detection and quantification of non-amplified nucleic acids. Direct hybridization of capture and detector oligonucleotides with the target allows sensitive and specific results in a timely fashion.

The present invention possesses numerous technical advances over the known art. For example, using the Electrochemical Gene Assay System, gene expression in cell cultures could be measured directly, without reverse transcriptase polymerase chain reaction ("RT-PCR") amplification. To measure gene expression, dual sensors can be incorporated into arrays in a micro-titer plate format. This format is widely used both in small biotechnology/biomedical research laboratories and in large, highly automated facilities for high-throughput assays in drug discovery and development.

All of the compositions, methods and apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. No

limitations are intended to the details of construction or design herein shown, other than as described in the claims below. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and apparatus and in the steps or in the segment of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the protection sought herein is as set forth in the claims below.