BBI Contributing Editor

SAN JOSE, California The annual Oak Ridge Conference, organized by the American Association for Clinical Chemistry (AACC, Washington), is the premier conference in clinical diagnostics for the presentation of emerging technologies that have the potential to shape the future of diagnostic testing. This year's conference, the 36th annual, explored a variety of technologies that may have a major impact on numerous segments of the clinical diagnostics market, including molecular pathology, nucleic acid diagnostics, immunodiagnostics, and point-of-care testing. A key theme for many of the research and development initiatives discussed at the conference was increases in the sensitivity of detection of clinically important analytes. Those advances promise to allow advances in the ability to detect diseases including cancer and infectious diseases at an earlier stage, when treatment can be most effective. Advances also were described in non-invasive and minimally invasive monitoring technologies, which may improve the management of diabetes and other chronic diseases.

Combining advanced high-sensitivity detection technologies with non-invasive or minimally invasive monitoring modalities holds the potential for significantly improving early disease detection. Examples of approaches described at the conference include highly sensitive and selective detection of markers of genetic diseases such as Fragile X Syndrome in prenatal testing, and detection of early markers of breast cancer using high-sensitivity analysis of breast fluid aspirates. A number of the development-stage technologies described at the conference are also expected to play important roles in the discovery of new disease markers, enabling advances in personalized medicine and pharmacogenetic testing.

New dimensions in pathology analysis

In the keynote address at the Oak Ridge Conference, Richard Caprioli, PhD, of Vanderbilt University School of Medicine (Nashville, Tennessee), described the use of imaging mass spectrometry as a discovery tool that may also have applications in the clinical pathology laboratory in the future. Caprioli described studies focusing on the spatial analysis of protein distributions in tissues using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The technique can be used to analyze large tissue sections (including sections of entire animals such as rats) at high spatial resolutions of about 25 microns to 30 microns, with the ability to measure up to 100,000 different biochemical species at each location (pixel) in the section, with a sensitivity of around one femtomole of protein per pixel. One application involves analysis of protein distributions in tumors to search for patterns of specific markers that may be used in diagnosis, prognosis and guidance of therapy. The technique can be applied to conventionally stained pathology slides, allowing correlations to be studied between traditional histopathology analysis and molecular marker distributions, and enabling the practice of molecular pathology at an unprecedented level of molecular resolution.

Caprioli has collaborated with Labcyte (Sunnyvale, California) to develop robotic processing systems used for slide preparation, since staining at 30-micron resolution would be impractical using conventional pathology techniques. Studies have demonstrated the capability to monitor protein patterns in embryo development, as well as in tumors, vs. normal tissues. In studies with human tumor tissue sections, Caprioli has found major differences between normal and tumor tissues for a particular patient but reasonable similarities between lung tumors from different patients. In addition, unique proteins have been discovered that appear in metastatic tumors but not in primary tumors, allowing the researchers to identify metastasis in sampled tissues that appear normal via standard histopathology, and potentially identifying markers of metastasis that can be used in therapy guidance. Correlations have been demonstrated between the existence of certain protein patterns and survival.

At present, the technology is far from mature and requires a pre-selection process to ensure that the quality of the tissue biopsy section is adequate. In addition, acquiring a high-resolution image of a typical large section can require a full day, although at medium resolution the acquisition time drops to one to two hours. Nevertheless, the high detection sensitivity (one femtomole of protein per 25 micron pixel) and high spatial resolution of the technique already is enabling biomarker discovery studies to be performed that allow tissue characterization at an entirely new level. Caprioli has begun investigating the potential to develop serum-based assays for proteins identified via tissue mapping for use in cancer diagnosis and monitoring.

Lori Zeller, PhD, of Lawrence Livermore National Laboratory (Livermore, California), described an even more powerful technology, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, that has the capability to perform similar tests but with an even higher mass resolution capability, expanding the range of molecules that can be analyzed. The technique can identify post-translational modifications of proteins, which occur before changes in protein expression according to Zeller, and could allow earlier detection of diseases such as cancer and genetic disease. Zeller is developing a Fragile X test that may be useful as a prenatal indication of ataxia/tremor syndrome which relies on the analysis of proteins in inclusion bodies, and also is evaluating a test that analyzes nipple aspirate fluid to identify markers of ductal carcinoma in situ.

Long-term, technologies such as imaging mass spectrometry could represent a next-generation approach to tissue and cellular analysis, allowing molecular characterization of biopsy specimens at a level of detail that eclipses the capabilities of today's methods many-fold. The market for automated cellular analysis products and reagents for performing cellular assays, as shown in Table 1, is exhibiting substantial growth at present, and is expected to expand to well over $1 billion within the next five years. The introduction of more advanced technologies is expected to drive continued growth in that segment of the global clinical diagnostics market.

A related segment of the cell analysis market that has also exhibited significant growth recently is chemoresistance testing for the guidance of cancer chemotherapy. Chemoresistance testing involves in vitro growth of tumor tissue biopsies followed by exposure of the cultured cells to various chemotherapy drugs to determine which drugs will be most effective in killing a tumor. At present, chemoresistance testing is performed by specialized reference laboratories that offer the tests to oncologists. The leader in the chemoresistance testing services market, Oncotech (Tustin, California), has experienced a 32% increase in the number of physicians requesting test services and a 16% increase in hospitals that submit specimens for testing within the past two years. Oncotech now provides testing services to more than 1,000 hospitals in the U.S. and Europe and has performed more than 85,000 tests using its Extreme Drug Resistance assay.

At the Oak Ridge Conference, a new chemoresistance testing technology was described that has been developed by Genoptix (San Diego, California). The Genoptix Optophoresis technology uses a moving optical gradient formed by a scanning near-infrared laser beam to quantify patient resistance or sensitivity to chemotherapy drugs. In contrast to assays such as the Oncotech EDR test, the Genoptix test does not require growth of the tumor cells. At present, Genoptix is focusing on testing of leukemia patients, since leukemia cells are very difficult to culture in a growth-based assay. The assay analyzes the cells' responses to the moving optical gradient, which depends on characteristics such as cell morphology, size, refractive index, density and surface properties, and assesses the correlation of those responses with apoptosis, or cell death, upon exposure to chemotherapy drugs. The assay can be performed with as few as 500 cells.

In a clinical study of nine patients performed in collaboration with the Scripps Cancer Center (La Jolla, California), the test results correlated with historical response in eight patients, and in the ninth patient a prospective outcome established by the test correlated well with clinical outcome. The company is establishing a CLIA-certified lab to begin offering testing services. Genoptix has received about $35 million in funding from a group of venture capital investors including Enterprise Partners, Tullis-Dickerson & Co. and Alliance Technology Ventures.

Researchers from Illumina (San Diego, California) and Veridex (Raritan, New Jersey), a Johnson & Johnson (New Brunswick, New Jersey) company, described a new gene profiling method that can be used to analyze archived pathology specimens to identify new tissue and cancer-specific markers. The method, which uses the GoldenGate assay system developed at Illumina, can be used to analyze formalin-fixed, paraffin-embedded tissues, which are the most widely available material for retrospective studies. Readout is performed with a 96-element fiber optic array matrix, allowing rapid and reproducible parallel analysis of well over 1,000 target sites. The method correlates well with other gene expression profiling techniques such as quantitative PCR.

Advances in nucleic acid diagnostics

A number of advances in nucleic acid diagnostics were described at the Oak Ridge Conference that will help drive continued growth in that segment of the diagnostics market, which already totals over $1.4 billion worldwide. James Prudent, PhD, of EraGen Biosciences (Madison, Wisconsin), described development of a new diagnostic platform that uses an expanded genetic code created by adding another synthetic base pair to the existing two-base pair code of native DNA. The added base pairs do not cross react with natural bases and increase the stability of DNA for complementary binding, but show similar destabilizing characteristics when mispaired. The technology has been applied in the Multi-Code assay to develop a genetic test for cystic fibrosis (CF). In comparison to the methods such as the Tag-It assay and the Roche (Basel, Switzerland) LightCycler assay, the Multi-Code procedure provides greatly reduced hands-on time (1.5 hours vs. 15 hours) and total assay time (4 vs. 32 hours), and eliminates the need for wash steps. In addition to CF testing, the technology also has been applied to platelet antigen screening. Although readout of the assay can be performed using either a solid-phase microarray or liquid chip (bead) format, the bead-based assay using the xMAP technology from Luminex (Austin, Texas) is the preferred technique, since it provides a wider assay dynamic range and entails simpler specimen handling procedures. A study comparing the Multi-Code CF assay to an existing FDA-approved test showed a 99.9% correlation.

Another molecular diagnostic assay using the Luminex 100 Liquichip system for readout was described by Abbott Molecular Diagnostics (Abbott Park, Illinois) and Celera Diagnostics (Alameda, California). The assay uses multiplex PCR to genotype 25 different SNP markers that provide a genetic risk assessment for venous thrombosis. The assay uses Oligonucleotide Ligation Assay for the genotyping reaction and exhibited a 100% genotyping accuracy in a study involving 1,260 patient samples.

Researchers from Corning's (Corning, New York) Life Sciences Division described the development of new microarray technologies that offer important advantages in cost, reproducibility, and sensitivity, and that have applications in both proteomics and nucleic acid diagnostics. Corning has built on its expertise in glass technology to create a new glass-on-glass array substrate with significantly (100-fold) improved binding capacity and a detection sensitivity of 25,000 copies vs. more than 400,000 copies with conventional arrays. The key feature of the new array is its use of a fritted glass layer that effectively converts the binding surface from a two-dimensional to a porous 3-D structure. The increased surface area provides enhanced sensitivity and dynamic range, and optimized assays using the Corning arrays exhibit excellent reproducibility of between 5% and 10%.

Diagnostic Products (DPC, Los Angeles, California), a company that until now has focused on the immunoassay market, described a development-stage nucleic acid diagnostic system using surface plasmon resonance fluorescence detection which eliminates the need for a wash/separation step in hybridization assays, while providing higher sensitivity than has previously been possible with homogeneous nucleic acid assays. When combined with PCR amplification, the prototype DPC system can detect a single copy of plasmid DNA. Improved sensitivity results in part from the use of a patented optical detection setup. A duplex assay for chlamydia and gonorrhea has been prototyped that uses standard specimen preparation techniques and requires only two minutes for the hybridization and detection steps. An evaluation of 265 specimens showed 100% sensitivity and specificity for chlamydia and gonorrhea, with the ability to detect doubly infected samples. In addition, a prototype Factor V Leiden assay is being developed. DPC said it believes the technology can be easily integrated into a fully automated nucleic acid testing system for high-throughput screening.

Cepheid (Sunnyvale, California) also is developing a new automated nucleic acid diagnostic system, the GeneXpert. The GeneXpert system uses real-time, multiplex PCR, and integrates PCR amplification and fluorescence detection in Cepheid's proprietary I-CORE module, a four-channel, solid-state, temperature-controlled fluorometer. Sample preparation is performed in a disposable cartridge containing dry PCR reagents that incorporates fluidics, filtration and ultrasonic lysis. The system is based on technology licensed from Lawrence Livermore National Laboratory. A GeneXpert assay for Group B Streptococcus was described by Cepheid researchers at the Oak Ridge Conference that will offer a 45-minute turnaround time and that can be performed at the point-of-care using the benchtop GeneXpert system. A pilot study evaluated the performance of the new assay on 49 patient samples vs. the IDI-Strep B assay, an FDA-approved nucleic acid diagnostic test for Group B strep that already is marketed by Cepheid, and found 100% concordance. Cepheid also has commercialized a non-automated nucleic acid diagnostic system, the SmartCycler, which uses the same amplification and detection technology used in the GeneXpert. The company has sold more than 1,400 SmartCycler systems since the product was introduced in May 2000. Cepheid plans to initiate clinical trials of the GeneXpert Group B Strep test soon. The GeneXpert system also is being developed for use in the Biological Detection System (BDS), a new rapid-response system for the detection of anthrax that will be deployed by the U.S. Postal Service. The anthrax assay detects four genetic targets simultaneously, including virulence factors and toxin-encoding genes, capsule production genes, an internal control, and a sample processing control, in order to achieve the goal of less than one false positive per 500,000 tests.

Another important application for rapid nucleic acid diagnostic testing is detection of Methicillin-resistant Staphylococcus aureus (MRSA). MRSA now accounts for up to 60% of all staphylococcus infections in U.S. hospitals. Nanosphere (Northbrook, Illinois) described development of a sensitive MRSA assay based on direct detection of genomic DNA using gold nanoparticle probes. The test uses Nanosphere's ClearRead microarray technology, which does not require the use of PCR amplification to achieve high-sensitivity detection, and the Verigene reader, which measures light scattered by the nanoparticle labels, which are 15 nanometers in diameter. The test procedure involves an initial 24-hour incubation of the patient specimen to produce culture isolates of Group B strep. After isolation, the procedure requires a 30-minute hybridization step, followed by a wash and a signal enhancement procedure. Less than 100 nanograms of genomic DNA is required to perform the test. While the current focus is development of a completely automated system for analysis of culture isolates, Nanosphere believes it will be possible to develop a future version of the system that does not require an overnight culture step.

An electrochemical sensor for DNA analysis was described by Dr. Jun Li of the NASA Ames Research Center (Moffett Field, California). While a number of electrochemical DNA sensors already are on the market, including devices from Nanogen (San Diego, California) and CombiMatrix (Mukilteo, Washington), their sensitivity is typically quite low compared to alternative technologies such as fluorescence. Li is developing advanced electrochemical sensors that rely on carbon nanotube arrays to reduce the effective surface area of the sensor, minimizing background and noise, while serving as highly sensitive sensing elements. To detect nucleic acids, a cyclic voltammetry technique is employed that relies on oxidation of guanine in the DNA to generate a current that can be correlated with the amount of DNA hybridized to capture probes attached to the nanotubes. The technique can detect nucleic acids at levels of a few nanomoles according to Li, or less than 1,000 molecules with a typical sensor. Li is now fabricating a multiplex genechip consisting of a 9-element array to allow rapid, low-cost detection of specific molecular targets such as pathogens and cancer markers.

Expanding the uses of immunoassays

Advances in immunoassay technology also were described at the conference that promise to expand the clinical applications of immunodiagnostics, including new proteomic assay technologies that allow simultaneous analysis of hundreds of analytes, vs. today's technologies that allow at most 10 to 20 analytes to be measured in a single assay. Approaches to increasing immunoassay sensitivity continue to be explored, which promise to improve the ability to detect diseases such as cancer and infectious disease at an early stage. John Russell of Abbott Laboratories (Abbott Park, Illinois) discussed a program to develop defined protein assemblies as diagnostic reagents. The technology involves engineering of layered protein structures by sequentially delivering activated components to a core protein immobilized on a solid support. The technique has been used to synthesize labeled antibodies for use in immunoassays that have up to 500 labels attached to each antibody, vs. five to 10 achievable with conventional labeling methods. The method can be used to synthesize antibody conjugates with enzyme, fluorescence and chemiluminescence labels. Furthermore, the number of labels per antibody can be precisely controlled, whereas with conventional methods labeling is heterogeneous. In actual practice, the degree of improvement is less than indicated by the increase in the number of labels due to non-specific binding of the antibody, and was determined to be about threefold in the case of a prototype chemiluminescence assay for thyroid stimulating hormone. However, Russell believes it will be possible with further applications of molecular engineering to reduce non-specific binding and realize a level of improvement that is closer to the theoretical limit.

Young Choi of Access Bio (Monmouth Junction, New Jersey) described a new supersensitive lateral-flow immunoassay platform using luminescent europium nanoparticle labels. The labels consist of between 30,000 and 2 million europium molecules encapsulated in a polymer capsule. The capsule is then coated with antibodies to the analyte of interest, and used as a second antibody that binds to the analyte after it has been captured on a nitrocellulose membrane coated with a first (capture) antibody. The technique provides a 30,000-fold amplification of fluorescence compared to labeling with fluorescein, and typically provides a 10-fold improvement in sensitivity as compared to colloidal gold labeling, a method often used in lateral flow immunoassay devices designed for point-of-care use. A prototype assay for the cardiac marker Troponin I was used to analyze serum, plasma and whole blood samples, and exhibited a detection limit of 25 pg/ml, close to that achievable with existing troponin assays available for automated analyzers used in the central laboratory. The assay format does, however, require the use of a fluorescence reader with a CCD camera detector, rather than visual readout. A highly sensitive assay for human chorionic gonadotropin (hCG) has also been developed by Access Bio.

Another fluorescence-based system described by researchers at Roche Diagnostics (Basel, Switzerland) uses a new time-resolved fluorescence energy transfer label allowing high-sensitivity homogeneous immunoassays to be developed. The labels consist of Ruthenium complexes with long fluorescence lifetimes that are used in combination with organic donor or acceptor dyes. The new Roche assay appears particularly promising for use in high-throughput screening assays because of its high sensitivity and low cost per test.

Perhaps the greatest increase in sensitivity for immunoassays, however, may result from using labeling methods that combine nucleic acid amplification with conventional labels such as fluorophores, dubbed Immuno PCR labels. PalindromX Group (Liverpool, UK) is one of the leaders in the development of those methods, and described results with a prototype TSH assay using its Melt Curve Analysis-Single Analyte Quantitation through Single Strand Extension (MCA-SAQSSE) technology. The method involves labeling of a detection antibody with a short DNA molecule (the "key") and, after binding of the antibody to the captured analyte, hybridization of a larger DNA molecule (the "lock"). The larger DNA contains a palindromic sequence (one that is identical in either direction, a configuration not occurring in nature) which can be amplified using PCR to produce a large number of fluorescence-labeled copies. The amplified product can be measured in real time using equipment such as the Roche LightCycler, now available in many molecular diagnostics labs. PalindromX has demonstrated at least a 400-fold increase in sensitivity as compared to conventional enzyme immunoassays in a prototype TSH test while maintaining precision of 5%. The company is seeking licensees to commercialize the technology for clinical diagnostic applications.

A number of companies described new clinical applications of high-sensitivity immunoassays at the conference. Ciphergen Biosystems (Fremont, California), a leader in the development of microarray technology for performing immunoassays, has developed a version of its ProteinChip that has applications in the early detection of ovarian cancer. The chip-based assay quantitatively measures six different markers, including three forms of transthyretin that result from post-translational modification, apolipoprotein A1, and an internal fragment of ITIH4, with a precision of about 10%. The assay is performed in a 96-well microplate format and requires about four hours to complete. The company believes that the measurement of different post-translationally modified forms of the target analyte may provide additional accuracy in the diagnosis of ovarian cancer.

Researchers from Pavlov's State Medical University (St. Petersburg, Russia) described an immunoassay for a new marker to detect epileptic seizures. Such an assay is particularly important for the diagnosis of epilepsy in young children, since EEG and clinical evaluation is only partially successful in detecting the disease. The NeuroTest-Epilepsy LA assay is a qualitative latex agglutination test that measures the GluR1 peptide, which appears in the bloodstream within one day after seizure-inducing agents are introduced into animals. In studies of 308 children and adults, the GluR1 marker exhibited 96% sensitivity, 94% specificity and 95% positive predictive value for the detection of epilepsy, making it a very valuable diagnostic tool considering that the positive predictive value of EEG alone is only 66% to 70%.

As indicated by the breadth of assay technologies under development, the immunoassay market has evolved to employ a wide array of labeling and detection modalities. As shown in Table 2, products using chemiluminescence labeling are exhibiting the most rapid growth in sales, followed by products that use fluorescence labeling. All of the top five suppliers in the global immunodiagnostics market now offer chemiluminescence-based immunoassay systems. The introduction of new immunoassay products based on advanced technologies is expected to expand the range of applications in immunodiagnostics over the next few years, driving substantial growth in the market.

New sensors to expand point-of-care testing

New point-of-care (POC) testing technologies were another important topic at the Oak Ridge Conference. Self-testing products for monitoring of blood glucose continue to dominate the market, but other growing segments include POC cardiac marker testing and coagulation self-testing. PortaScience (Moorestown, New Jersey) is one of a number of suppliers addressing the market for products used for self-testing of coagulation status, an area that has begun to expand as reimbursement has become available for certain categories of patients (primarily those with mechanical heart valves) who are on lifelong anti-coagulation therapy. PortaScience, a company formed about four years ago with a focus on POC testing products, has developed a portable device for the determination of prothrombin time, or international normalized ratio (INR), called the PortaPT. The device provides a visual readout of INR via a color bar from a 40 uL whole blood sample. The assay measures the distance blood moves along a capillary channel before it clots. A study comparing the PortaScience test to the ProTime POC INR system from the International Technidyne (ITC; Edison, New Jersey) unit of Thoratec (Pleasanton, California) found a correlation of r=0.94, and a working range of 0.8 to 5.1 INR units. Initially, the test is targeted for use by healthcare providers, but it is ultimately targeted to consumer use. It has the advantage of not requiring an instrument for readout, as do other currently available devices for coagulation self-testing such as the ITC ProTime, the Roche CoaguChek S, and the INRatio from HemoSense (San Jose, California). However, it currently lacks an on-board control, which is considered important for self-testing devices. PortaScience plans to offer the product at a transfer price of about $4, anticipating an end-user selling price of $10.

POC immunoassay testing is another major segment of the immunodiagnostics market, and has proven to be a particularly attractive area for suppliers of POC cardiac marker test systems. Biosite (San Diego, California), for example, reported a 68% increase in sales in 2003 largely due to increased revenues for its POC cardiac marker and BNP testing products. An initial user evaluation of the newest POC cardiac marker test on the market, the cardiac Troponin I test cartridge from i-STAT (East Windsor, New Jersey), a unit of Abbott, was presented by researchers from the Memorial Hospital & Health System (South Bend, Indiana). The i-STAT system offers the advantage of a broad menu of 16 tests including blood gases, electrolytes, glucose, creatinine and coagulation markers (ACT and PT/INR), along with Troponin I. Excellent agreement (R=0.94) was observed between the i-STAT troponin assay and the Dimension RxL troponin I assay from Dade Behring (Deerfield, Illinois), along with a 10% precision and analytical sensitivity of 0.02 ng/ml.

Another new technology for POC immunoassay testing was described by Future Diagnostics BV (Wijchen, the Netherlands). Future Diagnostics is developing a microfluidic device called the BioChip that incorporates all of the capabilities needed for performing an immunoassay on a single, disposable chip. Capabilities include micro sample aliquoting from an unmeasured specimen, automated reagent addition from on-chip reservoirs, computerized control of mixing and washing using miniaturized piezoelectric pumps, and detection by fluorescence resonance energy transfer. A prototype myoglobin assay has been developed with a sensitivity of 1 ng/ml, and sensitivity is about 100 times better than with a standard fluorescence immunoassay. The improved sensitivity is due in part to a feature known as kinetic sampling, which involves varying the flow rate of the reactants through the measurement zone depending on the concentration detected. At high concentrations, a high flow rate is used which avoids the need to dilute samples and re-run them as often occurs in existing immunoassay analyzers. At low concentrations, the flow is cycled back and forth through the measurement zone to provide increased sampling of the reaction, enhancing sensitivity. The company believes the analyzer can be reduced to the size of a hard cover book, and the disposable cartridge provides biohazard containment. Future Diagnostics plans to add multiplex assay capability and internal calibration to the next version of the system.

Another new sensor technology, with applications in rapid DNA and protein measurements at the point of care, was described by Peter Warthoe of Atonomics (Copenhagen, Denmark). Atonomics has developed a portable system that uses surface acoustic wave sensors to measure both DNA and proteins in the ng/ml to pg/ml concentration range. The goal is to develop a label-free sensor with a unit cost of 10 cents to 20 cents. A prototype sensor has been developed that incorporates an antibody to Troponin I along with an adjacent region that is coated with a material to correct for non-specific binding. A sensitivity of 2.5 pg/ml has been achieved in an eight-minute end-point assay, and Warthoe said he believes the sensitivity can be improved to one pg/ml by using rate analysis.

Many new developments in POC testing technology are focused on glucose self-testing, addressing a market opportunity that now exceeds $5 billion worldwide, as shown in Table 3. New technologies were described at the conference for non-invasive glucose monitoring, as well as for implantable, continuous glucose sensors. Hitachi (Tokyo) is developing a new non-invasive glucose monitor that uses the Metabolic Heat Conformation method. The method is based on the close relationship between metabolic oxidation of glucose in the body and body heat generation. Body heat generated by glucose oxidation depends on the balance of capillary glucose and oxygen supply to the cells, allowing blood glucose to be estimated by measuring body heat and oxygen supply, according to Hitachi researchers. The device employs six LEDS and two photodetectors to measure diffusely reflected light to determine blood flow and oxygen saturation, along with three thermistors and a thermopile to measure heat. In order to take a reading, the user simply places his or her index finger on a sensor pad, waits 10 seconds for equilibration, and reads the result on the portable instrument's display. A total of 18 optical measurements are performed per finger placement. The device must be calibrated for each user by a process that requires taking five to 10 capillary blood glucose measurements using a conventional blood glucose testing system over a period of a few hours, and fitting the data to a polynomial function. Population studies with the device have shown that patients can be divided into five clusters having different calibration characteristics. Additional development remains to be completed, including an assessment of the effects of fever and exercise on the readings. Hitachi is targeting launch in the U.S. and Japan sometime in 2005.

Sanford Asher of the University of Pittsburgh (Pittsburgh, Pennsylvania) described another approach to non-invasive glucose monitoring that relies on sensing of glucose and other analyte levels in tear film using photonic crystal technology. Photonic crystals are formed by charged polystyrene colloids that self-assemble into a crystalline colloid array, which is then embedded in a polyacrylamide hydrogel. The crystals diffract light with high efficiency, and the spacing of the crystal lattice determines the color of the diffracted light. To create a chemical sensor, molecular recognition agents are added to the colloid array that specifically bind to the analyte of interest, such as glucose. When the agent interacts with the analyte, the spacing of the lattice changes, producing a wavelength shift that can be measured to derive the analyte concentration. To create a practical glucose sensor, Asher's team has incorporated photonic crystals with glucose-specific molecular recognition agents into a contact lens, such that the sensor responds to the level of glucose in tear fluid via a wavelength shift. Readout can be performed using a simple device with a light source, mirror, and color comparison chart, allowing the user to illuminate the sensor and view the color of the diffracted light to determine glucose concentration within a narrow range. A company, Glucose Sensing Technologies, has been formed to commercialize the technology for glucose self-monitoring. Other applications under investigation include a creatinine sensor that can be used with urine, serum or plasma samples, and a subcutaneous sensor designed to respond to glucose or cancer markers that will be interrogated with near-infrared light. The latter project is being funded by the National Aeronautics and Space Administration.

An implantable continuous glucose sensor based on fluorescence detection is being developed by researchers led by Jerome Schultz, PhD, of the University of California, Riverside (Riverside, California). The sensor is constructed using a hollow dialysis fiber and includes an optical fiber that measures fluorescent-labeled dextran in the interior lumen of the dialysis fiber. Concanavalin A, which binds specifically to both glucose and dextran, is immobilized on the inner wall of the dialysis fiber. As glucose enters the interior of the fiber, it competes with the labeled dextran for binding to Con A, making the concentration of free-labeled dextran in the fluid within the fiber lumen dependent on glucose concentration. A fluorescence analyzer connected to the optical fiber allows continuous measurement of glucose levels in the region of the fiber. In animal studies, a 10 mm-long fiber with a diameter similar to that of a hair has been incorporated into a catheter probe, and good correlation with reference measurements of glucose concentration has been observed. Another version uses porous beads containing fluorochrome-labeled Concanavalin A entrapped within a hollow fiber. As glucose concentration increases in the medium surrounding the fiber, the labeled Con A is extracted from the beads into the lumen, and fluorescence increases. The fiber is implanted subcutaneously at a depth of about 0.5 mm, and interrogated with a fiber optic fluorometer through the skin to measure glucose levels in interstitial fluid. The sensor shows a linear response over the range of 0 mM to about 8 mM glucose in in vitro studies.

To expand the range of applications of the technology, Shultz is investigating fusion proteins as molecular recognition elements that exploit fluorescence resonance energy transfer as a sensing mechanism, including genetically engineered glucose responsive fluorescent proteins. The proteins can be incorporated within an implanted hollow fiber to create a continuous glucose sensor or using proteins that specifically recognize other targets, sensors for other analytes of interest for continuous monitoring.