An article on detection technologies that was previously published in IVD Technology’s 10th anniversary issue covered the more traditional means of developing assays that had already been commercialized and used for some time.1 That article also covered matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) technology in order to show some of the newer techniques for protein detection in human plasma and its use in tissue imaging (e.g., detection of cancerous tissues).

Figure 1. Continuous and simultaneous excitation and detection. Reproduced with permission from Pacific Biosciences.

This article will delve further into the development of MALDI-TOF in assays used for protein discovery. Several companies are involved in this area including Protein Discovery Inc. (Knoxville, TN), MDS Analytical Technologies (which was acquired by Danaher Corp.), and Bruker Daltonics Inc. (Billerica, MA). This article will also explore the latest developments in electrochemical detection and the commercialization of this technology for immunoassays and molecular diagnostics. In addition, this article will examine the concept of nanopore technology and its use in immunoassays, DNA detection, and DNA sequencing. In conjunction with nanopore technology, electrochemical detection is used to measures ion flow.

Single molecule detection involves many of the techniques that are mentioned above. It can be accomplished by a number of detection methods, including fluorescence, electrochemical detection, and surface-enhanced Raman spectroscopy (SERS). Pacific Biosciences (Menlo Park, CA) is working on commercializing this technology for DNA sequence analysis. Many universities are also involved in conducting basic research in this area, and commercialization of their efforts is not too far in the future.

A newer, label-free technology that is geared toward immunoassays and ligand binding has been launched on the IVD marketplace. This technology, known as bio-layer interferometry, was developed by ForteBio (Menlo Park, CA). This article will review the theory and application of bio-layer interferometry and its commercial impact. This article will also discuss the development and use of quantum dots.

Figure 2. Osmetech DNA diagnostic labeling system using ferrocene. Reproduced with permission fromOsmetech Diagnostics.

Single Molecule Detection
University laboratories have been investigating single molecule detection for the past 15 or more years.2-4 But not much of this effort has developed yet into IVD applications, which is a selling point for its commercialization. The single molecule detection technologies that are used tend to be confocal fluorescence, fluorescence based on double excitation, SERS, and electrochemistry (e.g., current, impedance). Some of these technologies have in fact been commercialized, and others will be commercialized shortly.

Single molecule detection by two photon excitation was discussed in 1995 and even in relation to photobleaching in 1998.5,6 The National Institute of Standards and Technology (NIST; Gaithersburg, MD) provided a tutorial on single molecule detection, but using a confocal microscope. An initial article on SERS pertaining to single molecule detection was published in 1997.2 But even though these studies showed that single molecule detection is feasible, not much to date has been commercialized.

One of the two single molecule detection techniques that is close to being commercialized is the use of a confocal microscope with single-mode waveguides (see Figure 1). Pacific Biosciences is using this technology in conjunction with labeled nucleotides to elucidate DNA sequence via DNA polymerase. While the concept is very simple, it has taken years to verify the proof of concept. With this technology, all of the chemistries are contained within a single-mode waveguide/orifice.

Figure 3. Voltamogram for the ferrocene system. Reproduced with permission from Osmetech Diagnostics.

A single strand of DNA is bound to the inside of the guide, and DNA polymerase is allowed to synthesize the complementary strand using nucleotides that contain a fluorophore. As the fluorophore is released from the guide area after each nucleotide addition, it is measured with fluorescence, and different fluorophore dyes are used for each nucleotide. If the system can measure each step along the way for each nucleotide addition, the DNA sequence can be determined. The basic system is shown in Figure 1. Of course the method has other applications (e.g., RNA, etc.), and its commercialization is expected soon.

Another single molecule detection method is the use of nanopores in conjunction with electrochemical detection. The nanopores may be created using inert materials or via protein layers on extenders. In this case, geometry, size, and charge are factors. The electrochemical detection technique may be the impedance of a straight current measurement. The build up of charges on a surface is the most commonly used method. Several IVD applications come to mind, in particular immunoassays. The addition of proteins on a surface leads to additional ions present, which leads to changes in impedance. Thus, this method can be used for diagnostic purposes. One of the ways is to capture specific DNA fragments, which results in this charge production. The capture can be accomplished via complementary pairs present, or specific proteins or peptides.

While several applications have been produced with these techniques, single molecule detection may be difficult to accomplish. But Oxford Nanopore Technologies Ltd. (Oxford, UK) has developed a material that can capture specific nucleotides upon their release. Coupled with electrochemical detection, this method produces specific single molecule detection. The company aligned this technology with DNA sequencing, and the release of a nucleotide produces a unique signal, which can then be observed.

Figure 4. Chemical Detection system used by CombiMatrix. Reproduced with permission from CombiMatrix.

Electrochemical Detection
During the past six years, two electrochemical detection methods have been commercialized. Both methods are microarrays, with detection that can be accomplished on microelectrodes. One method is the electrode system that was developed by Osmetech Molecular Diagnostics (Pasadena, CA) (see Figures 2 and 3). The DNA-based assay is performed on a chip with a multitude of assays. The labels are only present when a complementary duplex is formed. The reporter group is a ferrocene molecule that is attached to the DNA (or a protein molecule).

The system does not require a wash step to remove excess labeled single strands of DNA. Thus, after hybridization the readings are taken, scanning the voltage range produces a current which is then detected. Amplitude of the current is related to the amount of material present. The Osmetech system is commercially available and has been approved by FDA for three genetic diagnostic tests, including warfarin, cystic fibrosis, and a thrombophilia risk test. The tests are available in a cartridge format, and the liquids move throughout via microfluidics.

Another technology currently being sold is the electrochemical detection system by CombiMatrix (Mukilteo, WA) (see Figures 4-6). The concept is similar: assays performed on a microelectronic array system. The microelectrode is coated with a biological layer on which chemistries can be performed. Those chemistries may be DNA synthesis or placement of proteins on the surface by various means. The complementary DNA molecule may contain biotin molecules (as the protein) to which strepavidin labeled with HRP are added (this format may be altered).
The HRP consumes the substrate, which in turn causes a reduction of the oxidized substrate at the electrode surface. The current is then measured, and the amount of current that is observed is based on the quantity of HRP that is captured at the surface. The system, or current, is measured by the release of electrons from the capacitor, which is then bled to obtain a reading. The readout for a 12K chip takes 60 seconds. A protein assay for alpha 1-acid-glycoprotein (AGP) with a standard curve is shown in Figure 5. For the detection of spiked samples in a DNA sample, various concentrations of the spiked sample are presented in Figure 6.

Figure 5. Detection of alpha1-acid-glycoprotein (AGP). Reproduced with permission from CombiMatrix.

Other Detection Technologies
A newer detection technology, but with an old concept, that has been commercialized is bio-layer interferometry. ForteBio is selling this technology as packaged with a system called the Octet Instrument. The key benefits of this system are label-free detection, real-tine results, simplicity, rapidity, improves efficiency, and crude sample compatibility. Both immunoasaays and molecular diagnostics may be performed on this system. Applications for the system rely on kinetic characterization of the binding event. Protein quantitation in bioprocessing can be achieved with this system. The system can also be used for small and large molecules, and for assay development. This detection concept is shown in Figure 7.

An older detection method that has come into play is mass spectroscopy, specifically MALDI-TOF, although some newer techniques are being developed by Bruker Daltonics. The detection does require that the sample be isolated, and that can be accomplished by many means. The dried sample has a matrix added to it so that it enhances energy transfer from the laser to the molecule and causes it to be airborne. The charged sample is then passed through a field whereby the distance that is traveled is related to the size of the molecule. Often, there is a multitude of interfering substances, such as sodium dodecyl sulfate (SDS), which must be avoided. The matrices that are used vary, depending on the size of the protein, and some have been developed that can be used with a certain amount of SDS present.

Protein Discovery has developed a separation method that relies on electrophoresis, and the final product is collected and measured. In this manner, smaller proteins in plasma can be the focus of investigation for diagnostic assays. The components and changes in the plasma can be measured for proteins that are associated with disease processes, or for the detection of circulating cancer cell fragments.

Figure 6. Spike in control experiments. Reproduced with permission from CombiMatrix.

MS/MALDI-TOF is also being used to evaluate tissue slices of skin or organs in order to investigate abnormal cells that may be present.7 This process is currently being done as a service because of the high costs of the equipment. Bruker Daltonics is also using MS/MALDI-TOF, but its method varies a bit. In the case of imaging, the company calls the technology MALDI-IMS (imaging mass spectrometry), and for its example, Bruker studied the Her2 receptor for breast cancer. Of course, the more traditional mass spectrometry experiments are used for protein sequencing, carbohydrate analysis, and protein phosphorylation.

An area of research that has progressed toward commercial development is quantum dots. Nanomaterials of varying sizes are composed of silicon, germanium, and other semiconductor materials.8 Quantum dots produce varying emission colors that are based on their sizes. The smaller size emits a color in the ultraviolet region, while the larger dots emit a color in the red region. Researchers envisioned that quantum dots would surpass or supplant organic based fluorescent dyes.

The application of the quantum dot materials has found usefulness in biology, medicine, chemistry, and physics. In terms of immunoassay development, antibodies may readily be tagged with the quantum dot materials and used in performing traditional immunoassays. In this case, the assays for various analytes may be multiplexed because antibody specificity can be elucidated based on particle size.

More recent biological and medical applications have pointed toward imaging of biological cells. Again, the imaging will depend on the antibody tags that are used. The specificity will also depend on the antibodies that are used. This imaging may be done with live cells or in vitro imaging of organs in animals, such as mice. The use of nanomaterials in larger animals and humans will always result in extensive toxicological studies to ascertain the negative aspects that they may have on a living organism.9

Figure 7. Bio-layer interferometry. Reproduced with the permission of ForteBio.

References
1. A Kumar, B Anderson, and K Dill, “10th Anniversary Essay 4: Detection Technologies,” IVD Technology 11, no. 3 (2005): 58-61.

2. K Kneipp et al., “Single Molecule Detection Using Surface-Enhanced Raman Scattering (SERS),” Physical Review Letters 78 (1997): 1667-70.

3. J Clarke et al., “Continuous Base Identification for Single Molecule Nanopore DNA Sequencing,” Nature Nanotechnology 4 (2009): 265-270.

4. Y Astier et al., “Toward Single Molecule DNA Sequencing: Direct Identification of Ribonucleoside and Deoxyribonucleoside Monophosphate by Using an Engineered Protein Nanopore Equipped with a Molecular Adapter,” Journal of the American Chemical Society 128 (2006): 1705-1710.

5. J Mertz et al., “Single Molecule Detection by Two-Photon-Excited Fluorescence,” Optics Letters 20 (1995): 2532-2534.

6. C Eggling et al., “Photobleaching by Fluorescent Dyes Under Conditions Used for Single Molecule Detection: Evidence for Two-Step Photolysis,” Analytical Chemistry 70 (1998): 2652-2659.

7. HR Aerni et al., “High-Throughput Profiling of Formalin-Fixed Parafin-Embedded Tissue Using Parallel Electrophoresis and Matrix-Assisted Laser Desorption Ionization Mass Spectroscopy,” Analytical Chemistry 81 (2009): 7490-7495.

8. MA Walling et al., “Quantum Dots for Live Cell and In Vivo Imaging,” International Journal of Molecular Sciences 10 (2009): 441-491.

9. R Hardman, “A Toxicological Review of Quantum Dots: Toxicity Depends on Physicochemical and Environmental Factors,” Environmental Health Perspectives 114 (2006): 165-172.

Amit Kumar is chief executive officer at CombiMatrix Corp. (Mukilteo, WA). He can be reached at akumar@combimatrix.com.
 

Brooke P. Anderson is chief operating officer at CombiMatrix Corp. (Mukilteo, WA). He can be reached at brooke@combimatrix.com.
 

Kilian Dill is an independent consultant. He can be reached at redwoodranch@yahoo.com.