IVD companies have advanced considerably the ability to detect analytes in samples.
Amit Kumar, PhD, is president and chief executive officer, Brooke P. Anderson, PhD, is chief operating officer, and Kilian Dill, PhD, is director of intellectual property at CombiMatrix Corp. (Mukilteo, WA). The authors can be reached at firstname.lastname@example.org, email@example.com, and firstname.lastname@example.org, respectively.
In the early days, IVD detection technologies were predominantly optical methods and radioimmunoassays (RIAs).1,21, RIA was, and still is, one of the most sensitive assay methods available. However, the drawback of RIAs is their use of radiolabels. Another early mainstay was colorimetric and ultraviolet (UV) optical detection, or absorbance, including enzyme-linked immunosorbent assays (ELISAs).
An example of a clinical diagnostics system that utilized colorimetric detection is the Cobas Fara by Roche Diagnostics (Indianapolis). This system used a spinning rotor and colorimetric/optical assays for detecting analytes such as cholesterol, HDL, LDL, colorimetric iron, and serum proteins. Other IVD companies have used the spinning-rotor concept in conjunction with fluorescence detection for the quantitation of biomarkers associated with heart attacks.
A number of IVD detection products have also been based on reflectance and diffraction of material on a solid substrate, and visible-region optical- density experiments. Even today, such reflectance methods are still widely used in diagnostics. For example, the handheld monitor for HbA1c detection by Metrika (Sunnyvale, CA) uses a reflective method to obtain quantitation of HbA1c in blood.3 The technology enhancement for this device includes lateral-diffusion immunoassays along with a light-emitting diode and data collection. However, while quantitation is important, the reflective method does lack in sensitivity.
Another reflectance method that IVD companies, such as Biacore International (Neuchâtel, Switzerland), have commercialized is surface plasmon resonance spectroscopy. This method is a reflectance spectroscopy that makes use of the resonance shift from incident light and plasmon waves in the material upon binding of the molecules. The resonance shifts the reflectance maxima (angle difference), and the reflectance determines the quantity of binding on the surface. This technique does not require labels and is an ideal system for immunoassays, or binding-recognition events, and kinetic studies of the interaction of biomolecules.
Emission spectroscopy (e.g., fluorescence, phosphorescence, and luminescence) is a more sensitive detection technology that measures the emission of electrons gravitating from an excited state to a ground state. Because of quantum rules, an electronic state is coupled with a vibrational state. The emission is always found at higher wavelengths, and the frequency difference between excitation and emission is the Stokes shift. The greater the Stokes shift, the greater the ability to discern the two bandwidths. This principle is also true in luminescence spectroscopy, in which the emission may be a mixture of phosphorescence and fluorescence of metallic and organic dye bands (e.g., lanthanide emissions). In addition, fluorescence energy transfer (FRET), bioluminescence energy transfer (BRET), and scintillation proximity assays (SPA) are energy-transfer spectroscopies, in which light may or may not be emitted or quenched.4,5 To assist with these detection methods, Molecular Probes Inc. (Eugene, OR) and other IVD companies have developed compounds that can chemically attach to proteins or function as enzymatic substrates. The excitation of such compounds varies from the UV to the visible region. Many of the compounds also have appreciable Stokes shifts.
Figure 1. Cuvette-based fluorescence experiment. The light is administered to one face of the cuvette. Transmitted light is based on light not absorbed by the solution. Fluorescence is measured at 90 degrees from the administered light (click to enlarge).
Among the various emission methods, traditional fluorescence is the simplest and most widely used. From the simple quartz cell-based method of right-angle detection, IVD companies have developed a number of high-throughput systems (see Figure 1). Such systems are microtiter-plate-based formats in which emission is measured at angles much less than 90 degrees (e.g., epifluorescence). Companies such as Molecular Devices (Sunnyvale, CA) have developed assays based on fluorescence technologies which produced good results in drug binding and protein-protein interactions. With good sensitivity, the reactions may be monitored in real time, especially those involving enzymes. For the enzymatic systems, fluorogenic substrates are used so that the product formed is a fluorescent compound. Using enzyme labels as signal generators in immunoassays adds to the dynamic range for analyte detection.
Figure 2. The time-resolved fluorescence experiment. A light flash occurs at time zero, and fast fluorescence emission occurs early on. The sample-delayed emission occurs and may proceed up to 1 millisecond (depending on the sample). Data collection proceeds after 50 microseconds, and the fluorescence-integrated intensity occurs until 900 microseconds (click to enlarge).
Luminescence is a phosphorescence and fluorescence emission from a metal center such as lanthanide. Since luminescence involves an energy transfer, or intersystem crossing, from an aromatic antenna molecule to a metal center, the light emission is temporally resolved. For a complex metal system, the emission is often referred to as time-resolved fluorescence (see Figure 2).6 In this case, the fluorescence based on the metal center can be emitted milliseconds slower than the fluorescence based on the antenna molecule. Moreover, the Stokes shift is large, and each metal center has its own unique emission spectrum. Using this approach, many specific immunoassays may be carried out in one microtiter well, and the uncomplexed metal-chelated components can be washed away. Some IVD companies developed luminescence technology into products based on lanthanide-complex systems used in many hospitals. Analytes measured with luminescence included blood-serum proteins, phosphorylated proteins, Y. pestis, Enterotoxin B., and antibodies.
Figure 3. The fluorescence depolarization experiment. The sample is irradiated with polarized light. Light emitted is measured at 90 degrees from the original light source. Fluorescence is measured at both parallel and perpendicular directions. The extent of depolarization depends on the molecule's tumbling rate (click to enlarge).
Fluorescence polarization came out more than 10 years ago as a system design that utilized cuvettes or tubes (see Figure 3). Some IVD companies more recently developed a plate-reader format that was used in clinical assays as well as high-throughput drug screening. Fluorescence polarization uses polarized light to excite the label. The rate of subsequent depolarization depends upon the molecule's tumbling rate, which is affected by its structure and whether another molecule is bound to it. This temporal character of the depolarization of emitted light enables the distinction between small and large molecules as well as bound and unbound molecules. The advantage of this technique is the ability to monitor solution reactions of small and large molecules, such as ligand-receptor interactions. However, the sensitivity of this assay is reduced when compared with traditional cuvette-based fluorescence. Nevertheless, significant information has been obtained with this method for clinical assays.
Beads and Quantum Dots
During the past five years, several IVD companies have been dealing with bar coded beads, or different ratios of fluorescent dyes. Each bead contains a unique bar code that specifies the antibody or DNA sequence that is attached. An immunoassay may not be performed with the last piece of complementary DNA or antibody containing a fluorophore for detection. The bar code will establish which analyte or DNA sequence has been captured.
Quantum dots is another way to tag DNA or antibodies. Quantum dots excite at the same frequency but emit, or fluoresce, at unique frequencies depending on the size of the nanoparticle. Since various quantum dots conjugate to different antibodies or DNA, the analyte detected can be established by the emission frequency.
Figure 4. The FRET experiment. Two molecules contain very specific fluorophores that have an overlap in their emission and excitation bands (fluorescein emission with tetramethyl rhodium excitation). When the two molecules are bound, the energy transfer can take place resulting in emission energy associated with the TMR molecule (click to enlarge).
Energy-transfer processes, such as FRET, BRET, and SPA, offer other types of detection for diagnostic testing. In FRET, a molecule is excited and then transfers energy to a nearby second molecule (see Figure 4). The resulting energy transfer can result in emissions at much lower energy levels, or even quenched emission. In either case, the Stokes shift is large, and it is easy to distinguish between the emission from a first chromophore and a second chromophore. A FRET assay determines the proximity of a second molecule. Such information may be used to measure drug binding to receptors and other molecules. The assay may also be used in DNA-type work, in which one oligomer is closest to a second oligomer with a quenching molecule attached. The most notable of such assay systems are the molecular beacons, in which one end of a looped oligomer is conjugated to a receptor fluorophore and the opposite end is attached to a quencher molecule. In the molecular beacon assays, the fluorophore is inactive until it is hybridized to a complementary DNA strand and the loop becomes linear. This separates the two fluorophores and allows emission to occur.
BRET begins with a biofluorescent protein, such as green fluorescent protein (GFP). This protein may absorb light and perform a transfer of energy (e.g., radiationless, dipole-dipole) to a biofluorescent molecule that comes in close contact or is bound to the GFP. The measurements and parameters that provide information about a ligand binding to a receptor or a drug molecule binding to an active site are similar to FRET.
SPAs are the same as the two energy-transfer processes described above. In SPA, a fluorophore emits high energy upon contact with a radiolabel. The addition of a competing ligand may inhibit the radiolabel compound and reduce the emission of light.
Electrochemiluminescence is a non-destructive detection technique that utilizes an electrochemically activated molecule as a transfer mechanism of energy to a metallic ruthenium-based complex. The complex emits light in a luminescence process and is conjugated to one of the binding molecules like an antibody. Igen International Inc. (Gaithersburg, MD) licensed the electrochemiluminescence technology to Roche for developing the Elecsys system.7 The system is based on immunoassay detection methods, in which ruthenium labels need to be introduced. Diagnostic applications of this system include the detection of serum proteins such as thrombin, beta-lactam antibiotics, hormones, and food-borne pathogens.
Certain methods can detect small quantities of infrared emissions from samples. Such methods were originally used to obtain information in glucose monitoring. However, that concept has not yet been widely commercialized. Some glucose monitors in the latter stages of development do use infrared emission to measure glucose in tissue or serve as heat sensors. The heat sensors detect differences in an organ due to attached labels or slight temperature differences due to metabolism. Such sensors may aid in discovering tumors or malfunctioning organs.
Electrochemical detection is used to detect minute quantities of glucose. In this method, a few microliters of blood are required, and glucose oxidase is used as the enzyme that oxidizes glucose and generates an electrochemical signal.8
One of the major breakthroughs in proteomics has been the development of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) technology for protein identification and detection. Although this technology has been around for 15 years, recent modifications and applications have made MALDI-TOF an up-and-coming tool that relies upon mass spectrometry for detecting protein samples in serum, body fluids, cell products, saliva, etc.
Mass detection is based on the macromolecules' flight time. Protein molecules must be separated from the complex composition by using techniques such as enzymatic digestion. A matrix of sinnapinic acid in acetonitrile, containing water and trifluoroacetic acid, is added to the protein sample. The matrix mix is allowed to dry and is placed into a mass spectrometer. A laser brings the sample and matrix into the gas phase. Some of the sample undergoes protonation or deprotonation to produce a charged protein sample that travels toward the detector. The time of flight then gives mass values for the protein.
Experiments have shown that serum components can be identified, leading to the detection and verification of biomarkers for diseases. With the identification of such biomarkers, drug compounds can be developed and studied.
Mass spectrometry can also investigate the behavior of organs and the metabolites produced from diseased and healthy states.9 The metabolite-marker studies can then locate demarcation lines of the disease process within organs.
Figure 5. The redox enzyme amplification scheme used in a detection platform by CombiMatrix Corp. (Mukilteo, WA). The conversion of substrate to product by the redox enzyme (horseradish peroxidase), produces an electron flux. It is the electron flux that is measured as a current (click to enlarge).
IVD companies have shown great interest in developing assays based on biochips. Biochips may be produced by spotting or direct syntheses on the material or substrate, using photolithography, electrochemistry, or ink-jet technology. While the material placed on a chip is usually DNA, other materials can be used, such as RNA, peptides, proteins, and carbohydrates. Fluorescence is the most widely used detection method in biochips. However, recent work by CombiMatrix Corp. (Mukilteo, WA) has demonstrated that redox-amplified electrochemical detection is more sensitive than traditional fluorescence methods and is less expensive (see Figure 5).
DNA chips can determine gene expression, single polymorphism analysis, and DNA content. In December 2004, the Cytochrome P450 chip by Affymetrix (Santa Clara, CA) became the first DNA chip to receive FDA approval for clinical diagnostic use.
IVD companies have made great strides in developing detection methods during the past 10 years. Optical methods are being redefined and still provide a viable technology. At the same time, the advent of microarrays and electrochemical detection, as well as the expansion of older technologies such as MALDI-TOF, will contribute to the further development of detection technologies in the future.
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