An emerging technology using fiber optics has potential as an alternative in platforms for clinical diagnostics.
In efforts to provide alternative test platforms for the clinical environment in both centralized and decentralized settings, large and small IVD companies alike continue to invest significant finances into the research, development, and production of innovative diagnostic technologies. Although the majority of such technologies are primarily modifications or adaptations of current systems, some are indeed truly pioneering.
Figure 1. Prototype of the Luna long-period grating detection system. The system employs optical-fiber sensors that can be multiplexed by using a different sensor for each single-analyte measurement.
In recent years, the most important trends in improving IVD test systems have been to make the platforms more sensitive, more accurate, portable, and cheaper. In many cases, companies have realized that such improvements could be accomplished by adapting or merging very different technologies from other research fields for IVD use. Examples of adaptations of traditional technologies include the gradual development of basic detection systems employing spectrophotometers. Such devices utilize general chemistry principles to detect a color change that permits the quantitation of analytes, and in turn have led to the development of very sophisticated miniaturized systems. The latter include plate readers for enzyme-linked immunosorbent assay (ELISA) testing, which utilize the same chemistry principles in combination with antibodies to evaluate analyte concentrations at the nanogram level or lower. Devices based on such simple biochemical detection principles are currently in use in both the central laboratory (where they take the form of automated batch analyzers) and in near-patient systems.
The mode of detection used by such instruments may be chemiluminescence, fluorescence, radioimmunoassays, or DNA microsensors. The last of these has evolved significantly during the past decade to meet the demands of genomics companies. Indeed, progress in this area of scientific research has been so rapid that the human genome project was completed in 2000, taking a decade less than originally expected.1–3
The use of electrophoresis, which separates charged molecules by means of an electric field, has also evolved rapidly over the past two decades. Where large, cumbersome gels with poor resolution and lengthy separation times were once the norm, electrophoresis can now be performed using minisystems. In recent years, electrophoretic instrumentation has been adapted for use in a variety of clinical settings. For example, such instruments can be used to measure proteins such as creatinine kinase (CK), CK-MB, and the MB isoforms that are released as a result of cardiac tissue death.
The objective of this article is to describe how certain companies are adapting and merging innovative technologies to further improve their diagnostic platforms for use in clinical settings. Some of these systems are considered pioneering, and will be discussed in some detail. The article will also provide an overview of a potentially useful detection system that utilizes long-period grating (LPG) fiber optics.
Limitations of Traditional Technologies
Although the aforementioned technologies are in routine use in clinical settings, instrument platforms based on such technologies often have operational limitations that make them unsuited for some clinical applications. For instance, spectrophotometers, the basis of many IVD systems, use quantitation of color change to evaluate the presence or absence of analytes in tissues. However, spectrophotometry-based platforms can have less sensitivity and specificity than systems using other technologies, and can encounter difficulties when certain interfering substances are present. Such interferences include lipemic, hemolytic, and icteric samples. Consequently, manufacturers seeking to minimize the need for extensive sample preparation by designing systems that can analyze whole-blood samples have begun to shift to technologies such as chemiluminescence, which can offer greater sensitivity and resistance to interferences.
Figure 2. Diagram of an LPG sensing platform showing the optical fiber with grating and the demodulation support system. Light traveling through the optical fiber is scattered out of the grating at different optical wavelengths. The index of refraction of the coating changes due to target-molecule absorption and, when demodulated, provides a quantitative assessment of analyte concentration.
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Similarly, electrophoresis and ELISA technologies are not suited as platforms for use in near-patient settings. Both of these technologies are routinely used to diagnose chronic, pathological conditions. But in acute-care settings these systems are not appropriate. For proper analyte quantitation, they would require the employment of densitometry or cumbersome plate readers. Hence, manufacturers are involved in an ongoing quest to optimize assays for patient triage in acute clinical settings.
One of the current trends in IVD technology is the development of systems for the quantitative determination of multiple analytes in a single specimen.4,5 Such multiplexed assay systems have the potential to improve patient outcomes by providing information that enables healthcare professionals to triage patients with regard to the stage of their disease.6,7 This is especially important for diseases with pathological continuums. One example is myocardial infarction, where the choice of therapeutic intervention—typically either thrombolytic therapy or percutaneous transluminal coronary angioplasty—can be very different depending on the stage of the disease.8
However, there are major limitations to optimizing multiplexed assays. Such limiting factors include cross reactivity from ‘like proteins’ that may be recognized as pathological. For example, an immunoassay that measures d-dimer protein—a breakdown product of blood clots that is considered a marker for both deep vein thrombosis and pulmonary embolism—may also cross react with fibrinogen—a naturally occurring and circulating coagulation blood protein important for normal hemostatic function. Immunoassays used in multiplexed systems therefore need to be optimized to ensure negligible cross reactivity.
Emerging Innovations in Diagnostics
Figure 3. Optical-fiber LPG-based chemical and biochemical sensing platform for monitoring physiological targets.
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Traditionally, one of the major challenges in developing innovative platforms for IVD testing has been bridging the gap between the engineers who develop the test platform and the biochemists who formulate the chemistries in order to attain optimal analyte interaction and detection signal. Many companies recognize that in order to optimize clinical diagnostic platforms, it is important for these groups to work closely together. Without a cooperative effort from these disciplines, platform development timelines are prolonged with consequent delays in investment recuperation and improved patient care.9–11
One company that has recognized the importance of such interdisciplinary interaction is Luminex Corp. (Austin, TX). By ensuring that its engineers understand the clinical objectives, Luminex has developed the LabMAP system, which operates in conjunction with the Luminex100 analyzer to multiplex up to 100 simultaneous diagnostic tests on a single specimen.5,12 This system uses polystyrene microspheres that have previously been impregnated with two fluorescent dyes. Both dyes can fluoresce at ten intensity levels. By illuminating the sphere with one laser, the ratio of the two dyes identifies the sphere set. A second laser simultaneously excites a surface fluorophore to quantitate the specific assay being tested.
Figure 4. Dose response curves of affinity LPG sensor for detection of the HIV p24 protein. Both direct (rabbit detecting antibody) and sandwich (goat antirabbit IgG) are shown (additive wavelength shift from baseline ~x7).
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Current clinical applications of the LabMAP technology include allergy testing, which is considered semiquantitative, and screening for abnormal coagulation proteins, such as Factor V Leiden, as well as autosomal recessive genetic disorders, including cystic fibrosis.5,12
The Biacore platform by Biacore (Upsalla, Sweden) utilizes a surface plasmon resonance (SPR)–based biosensor technology to monitor biomolecular binding in real time and label-free mode. In addition to not requiring purification of the target molecules prior to analysis, with this system the ligand of interest is bound directly to a dextran-coated gold layer by a predefined covalent or affinity linkage. The ligand is freely accessible to three-dimensional biomolecular interactions that are measured directly by monitoring changes in the SPR signal. This signal is a reduction in the reflected light intensity that is measured as a component of both angle and wavelength. Applications of this technology include sample screening for drug discovery, antibody isolation for diagnostic and therapeutic use, and monitoring vitamin content in food.
Another example is the IAsys platform from Thermo Labsystems (Helsinki, Finland). This platform resulted from a collaboration between Thermo Labsystems, GEC-Marconi (London), and the Institute of Biotechnology (Cambridge, UK). Available in a range of models from near-patient testing to fully centralized units, IAsys has many of the features that are desired in clinical chemistry analyzers, including flexibility and user-friendly operation. As with the BIAcore technology, IAsys analyzes biomolecular interactions in real time without the need for labels or purification. These systems use an innovative resonant mirror technology, which has demonstrated greater test sensitivity than comparable systems. This technology has multiple applications, including serum or other fluid analyte determination, concentration monitoring, hormone receptor binding studies, and cell recognition and histocompatibility evaluation.
All of the previously mentioned systems have current clinical applications and strive to overcome limitations that are associated with other detection technologies. Moreover, they all lend themselves well to high-throughput screening and as such have the potential for clinical use for genetic screening. Although these systems in their current formats may not be suitable for near-patient testing, their clinical applicability will undoubtedly continue to increase.
Long-Period Grating (LPG)
Luna Analytics (Blacksburg, VA) has also managed to integrate its engineering and biochemistry personnel to develop fiber-optic sensors that can evaluate chemicals such as toxins and biowarfare agents. This process is accomplished by etching a grating into a fiber that experiences color changes when it is in the presence of the molecules being evaluated. These color changes may then be quantitatively analyzed by a downstream detector.
Figure 5. Sensorgrams demonstrating direct and sandwich-assay detection of HIV protein p24. Once an initial baseline signal is obtained (dotted line), two different p24 concentrations, 5 ng/ml (a) and 1 ng/ml (b), are added and a new baseline (B) is established. The next two peaks show the effects of binding antibodies and the new baselines that arise. The sensor is regenerated by washing with thiocynate (SCN), thereby restoring the original baseline.
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Optical-fiber sensors have a history of success in a number of applications and may be used in certain clinical settings for both diagnostic and therapeutic applications. They offer an alternative approach to obtaining biological, physical, and chemical measurements in various environments. In addition to IVD applications, for example, with minimal modification and sensor configuration the fibers can be multiplexed in large numbers along a single fiber transmission cable for in vivo use to detect a variety of target molecules. Some of these biochemical targets have already been detected using the Luna technology.13
The Luna system utilizes an optical LPG platform that detects index-of-refraction changes. These changes result from the rapid binding of target molecules that are selectively captured by affinity coatings applied to the optical fiber. The LPG technology is capable of detecting extremely low concentrations (1 x 10–12 mol) of target analytes. The Luna system comprises two primary elements: the LPG affinity-coated sensing element and a demodulation system (see Figures 1 and 2).
The Luna LPG is a spectral-loss element that scatters light from an optical fiber at a predetermined wavelength that depends on the grating period (see Figure 3). The grating is located within the core of the fiber, which is composed of germanosilicate glass. Once exposed to ultraviolet light, a permanent and measurable refractive change is created. In addition, the light scatter depends on the refractive index of the fiber and its surrounding environment.
LPG-based biochemical sensors operate with specifically developed affinity coatings or swellable polymers that permit selective, quantitative changes in the refractive index when target analytes are present. Upon adsorption, the refractive index surrounding the LPG region changes in accord with the spectral shift in the wavelength of scattered light that correlates to the amount or mass of bound material. This change is then demodulated to determine analyte concentration.
The operating wavelength can use different grating periodicities, and the LPG sensors can be written at various wavelengths for simultaneous demodulation using standard wavelength-division multiplexing techniques.14
Figure 6. Dose response curves of LPG sensors in evaluating matrix effects when the HIV p24 protein was spiked into human sera and buffer.
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The LPG device is embedded in an optical fiber with a diameter of 125 µm and a sensing region that is 1 cm long. The sensor is used for reflection so the sensing region is at the end of the optical fiber. Depending on the size and flexibility of the device, the LPG can be used in a flowcell or in a direct-sampling configuration, such as a vacutainer, or can be embedded as a subcutaneous device. Since multiple sensing regions can be incorporated into a single optical fiber, panel testing can be conducted on a single small-volume sample.
This technology is not based on absorption or on determining spectral content. Therefore, it is not susceptible to interferences from endogenous materials that are typically found in serum, urine, or whole-blood samples.
To perform a test, the operator places a sample in the sample queue and initiates the instrument. The sample is accessed by the instrument through a robotic handler and introduced to the LPG sensor, which is located in a single-use cartridge.
Sample processing occurs through the instrumentation platform and consists of incubation and wash steps. The signal-conditioning system then interrogates the sensor and determines the changes in optical signature caused by binding of the target. The changes in signal correspond to concentration. After the test is completed, the self-contained cartridge is discarded, and the sample container can be retained for additional testing.
The current sensor format has been designed and evaluated for proteomics with a reuse lifespan of approximately 100 tests. However, the next generation of sensors will be designed as a single-use disposable for detecting multiple analytes.
Applications of LPG Sensor Technology
The Luna fiber-optic technology offers several approaches to making biochemical measurements in clinical settings. The applicability of LPG and optical-fiber sensors for multiplexing numerous tests continues to be expanded and is another example of the evolving trends in the IVD sector.
Figure 7. Schematic of the optical fiber of an LPG-based affinity sensor and detection scheme used to demonstrate ß-galactosidase detection.
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Initially developed to measure physical properties, including strain, temperature, pressure, acceleration, refractive index, force, skin friction, viscosity, and magnetic fields in industrial sectors, the Luna sensors have recently transitioned into the clinical arena. The transition to clinical settings involves coating the LPG sensor with specially designed affinity coatings, including monoclonal antibodies, in order to absorb selectively the target molecule and provide real-time monitoring of disease pathogens.
Clinical applications that have already been tested using the LPG system include drug discovery, diagnostic applications, and point-of-care (POC) testing. One of the most innovative applications of this technology has been for screening combinatorial chemistry libraries, thereby permitting researchers to access large arrays of candidate drugs and accelerate the discovery of new pharmaceuticals.
LPG biosensors have also been used for detecting human immunodeficiency virus (HIV) proteins at titers less than 1 ng/ml and for studying other protein-protein interactions. In addition to the concentration of HIV proteins, overall viral load may be quantitated from the calibration curve. Once virus size has been determined, the viral load may be extrapolated from the calculated molar ratio.
In one set of experiments, different concentrations of the p24 HIV protein were quantitated by using monoclonal antibodies coated onto the biosensor fiber and by plotting the coupled wavelength, which is related to analyte concentration versus time in sensor-grams (see Figure 4). In essence, these measurements represent the response of the sensor versus time. The wavelength is a direct index of reactive index or bound mass that is a characteristic of the individual fiber profile and grating period.
For this series of tests, measurements were performed in multiple fibers and tested in triplicate. Following baseline acquisition, the target analyte was added to the sample, a wash step removed any nonspecifically bound substances, and a new baseline was established. The difference in wavelength is directly proportional to the concentration of bound analyte.
Figure 8. Response of LPG-based biosensor to ß-galactosidase and anti-IgG antibodies.
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Amplification of sensor response may be achieved by increasing the bound analyte mass and using additional antibodies in sandwich formation (see Figure 5). Analyte concentration can be determined within five minutes, which makes this technology useful in POC applications. One example shows the dose response curves of the Luna affinity-LPG sensor for three different concentrations of the p24 HIV protein (see Figure 6). These data from a matrix evaluation experiment show the p24 HIV protein spiked into human sera, compared with a spiked buffer.
LPG sensors have also been used for monitoring microbial activity. For these tests, the sensors were coated with an affinity purified anti-b-galactosidase antibody. After the sensors are exposed to the samples and washed to remove unbound material, the increase in wavelength is represented by microbial activity that is determined by the concentration of bound b-galactosidase (see Figures 7 and 8). Test samples included b-galactosidase (10 µg/ml), as well as positive and negative controls which were employed to demonstrate that rabbit anti-b-galactosidase antibodies were on the fiber (red bars) and the specificity of the rabbit IgG (purple bars). The target analyte (b-galactosidase) produced a wavelength shift of ~0.6 nm, while the positive control produced a wavelength shift of ~2.8 nm. This technology can be expanded to utilize specific microbial components such as membrane antigens, capsular antigens, and antibodies for monitoring both gram-negative and gram-positive bacterial activity.15
Other potential clinical areas where the Luna LPG may be used in the near term include cardiac catheterization and gastrointestinal disease. These sensors may also be used for interventional cardiology to determine the level of factors, or proteins, of the coagulation cascade, which can assess a patient’s risk of subsequent coronary occlusion or pathological bleeding periprocedurally. Moreover, current methods for diagnosing colorectal cancer include sigmoidoscopy, which utilizes fiber optics to visualize cancerous polyps in the lower bowel, or colonoscopy, which is expensive, uncomfortable, and rarely covered by insurance. LPG sensor technology using fibers coupled with high-affinity antibodies may allow triage of the stage of the disease at the site. Although current sigmoidoscopy procedures may involve fiber optics, multiple fibers that are specific to certain receptors expressed at different stages of the disease may be used for diagnosis and thereby permit the appropriate and optimal delivery of anticancer agents.
Colonoscopy is a more invasive procedure that evaluates larger sections of the bowel and carries the risk of possible complications, including bowel perforation. The utility of a multiplexed fiber-optic system with therapeutic potential may offer a more reliable alternative to current techniques.
While numerous celebrities have promoted heavily the use of fiber optics in the telecommunications industry, significantly less is known about the expansion of this technology into other sectors, including biomedical and clinical applications. The Luna platform combines affinity adsorption, using an array of independent elements that provide accurate analyte quantitation, with sensitivity at the parts-per-trillion level. Furthermore, Luna has incorporated this technology into a unique system design that comprises a compact, lightweight biochemical detection platform suitable for both centralized (lab) and decentralized (point-of-care) clinical settings.
Further development of the current configuration may also have potential applications as a research tool for drug-discovery screenings. Using the multivalent optical sensor design, this system may be expanded to more than 100 independent target signals with a total assay cycle time of approximately five minutes.
What is also apparent in the Luna and other emerging platforms is that their clinical utility will continue to expand. With the current clinical drive to optimize and multiplex genetic screening and to aid in the monitoring and management of disease with multiple etiologies, the adaptation of current detection modalities to alternative platforms is worth considering. The trend toward a closer working interaction in all aspects of product development between the chemists and engineers should make this transition a smooth one.
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David G. M. Carville is president of Causeway Scientific (Mishawaka, IN). He can be reached via firstname.lastname@example.org.
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