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Clinical Chemistry 45: 453-458, 1999;
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(Clinical Chemistry. 1999;45:453-458.)
© 1999 American Association for Clinical Chemistry, Inc.

Review

Nucleic Acid Detection Technologies — Labels, Strategies, and Formats

Larry J. Kricka

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104.


   Abstract
Top
 Abstract
Introduction
 Labels and Sensitivity
 Convenient and Simple Assays
 New Challenges for Nucleic...
 Conclusions
 References
 
Currently, no consensus exists on assay formats, labels, or detection reactions for nucleic acid assays. New labels continue to be developed and tested, and recent candidates include acetate kinase, firefly luciferase, and genes for enzymes. An additional trend is toward nonamplification strategies (e.g., branched chain and dendrimer type assays) as alternatives to the popular PCR and related amplification strategies. The new wave of microanalytical devices (microchips, with nanoliter to microliter internal volumes), massively parallel simultaneous test arrays, and the desire to produce hand-held sensors present new challenges and requirements for nucleic acid detection methods (e.g., analysis of large arrays of micrometer-sized spots of nucleic acid with high resolution). Here I review selected developments and new directions in nucleic acid assays.© 1999 American Association for Clinical Chemistry


   Introduction
Top
 Abstract
 Introduction
Labels and Sensitivity
 Convenient and Simple Assays
 New Challenges for Nucleic...
 Conclusions
 References
 
The range and scope of nucleic acid-based assays continues to expand (e.g., medical, forensic, and environmental applications) (1)(2)(3). Many of the types of nucleic acid assays developed require a secondary detection technology, e.g., a label, because a nucleic acid does not have intrinsic properties that are useful for direct high-sensitivity detection. As yet there is no consensus on the choice of a label for nucleic acid detection, and none of the current labels has the attributes of a universal or ideal label. Key factors governing label choice include stability, sensitivity of detection, speed and convenience of detection, and the overall cost for the label, detection reagents, and detection system. In addition, modulation of a property of a label when a conjugate binds to a binding molecule is also desirable because it can form the basis of nonseparation assays. Two new analytical formats, the microchip and the microarray, pose new challenges for nucleic acid detection (4)(5)(6). Here I review current directions in nucleic acid analysis and discusses recent strategies for ultrasensitive detection (Table 1 ).


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  		Table 1. Selected labels and strategies for nucleic acid detection.


   Labels and Sensitivity
Top
 Abstract
 Introduction
 Labels and Sensitivity
Convenient and Simple Assays
 New Challenges for Nucleic...
 Conclusions
 References
 
The level of interest and the rate of progress toward ultrasensitive detection methods is apparent from a simple search of the medical literature database (Table 2 ). The first citations for analytical methods with detection limits at the zeptomole (10-21 moles) and yoctomole (10-23 moles) do not appear until 1991 (7)(8). In the analytical literature, there are earlier studies using more complex techniques that achieved single molecule detection limits (i.e., 1.7 yoctomoles) (9), but these methods were not suited to routine clinical laboratory applications. Several different strategies have been developed for ultrasensitive assays, and these can be broadly categorized into the use of (a) improved labels, (b) multiple labeling, and (c) background noise reduction.


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  		Table 2. Growth of ultrasensitive analytical techniques.

improved labels
Most if not all nucleic acid assays are a sandwich format and exploit the specificity of base recognition (adenine for thymine, guanine for cytosine) and the high binding constants of the resulting duplexes. Label detection is a key determinant of sensitivity for sandwich assays (10). Thus, overall assay sensitivity can be improved by using a label that can be detected with higher sensitivity. One such label is acetate kinase. This enzyme label can be detected down to 8.6 zmol using a coupled bioluminescent assay. ATP formed by action of the enzyme label on an acetyl phosphate substrate is measured using the firefly luciferase reaction. Initial data from a blotting assay for transferrin revealed a detection limit in the low nanogram range (11). A beneficial feature of this enzyme label is that the bioluminescent light emission is a stable glow (>100 min), and this is advantageous for signal acquisition using an imaging device, e.g., for nucleic acid blotting applications.

In addition, the expressed enzyme and the gene for the bioluminescent luciferase class of enzymes have considerable potential as labels for nucleic acid assays. The detection limit for this enzyme is excellent (7 fg, or 0.1 amol) (12). Furthermore, manipulation of the gene permits the design of mutants that have improved thermal stability and also mutants that catalyze light emission at different wavelengths (13)(14). Modification of residues of firefly luciferase at positions 215 and 345 produces the double mutant, A215L + E354K, and this molecule is stable for prolonged periods (retains 50% activity after 5 h at 37 °C) (12). Even greater improvements are obtained by mutation of the Luciola lateralis luciferase. The mutant Ala217Leu retains >70% of initial activity after 1 h at 50 °C (15). Random mutagenesis of the luciferase gene from Hotaria parvula produces mutants that have a red-shifted bioluminescent light emission (maximum emission at 610 nm vs 565 nm for the wild type) (13). This is advantageous because it offers a multiplexing strategy for simultaneous assay of multiple analytes that would use a common firefly luciferin-based detection reagent. Results of preliminary experiments with the double mutant chemically attached to a thiol oligo-dG using a maleimide-based bifunctional coupling agent are encouraging, but the conjugate did lose luciferase activity (12). This should be resolvable by exploitation of an additional benefit of the luciferases, namely that fusion conjugates can be made that preserve the enzyme activity of the luciferase and the molecular recognition properties of the binding molecule (e.g., firefly luciferase-Protein A, Luciola luciferase-biotin acceptor molecule, and luciferase-RNA binding protein) (15)(16)(17)(18). This genetic engineering approach to conjugate production offers a means of ensuring a reproducible supply of fully active conjugate for assays. Instead of using the product of a gene as a label, an alternative strategy is to use the gene itself, and in particular the gene for an enzyme, such as luciferase (19)(20)(21). In this way, a very high amplification factor can be introduced into the assay because many enzyme molecules can be transcribed from each individual gene label and each enzyme catalyzes the formation of many product molecules per minute. The initial success with the firefly luciferase gene label in immunoassay has been extended to DNA hybridization assays. Model assays detected as low as 0.1 fmol of target DNA (signal-to-background ratio of 2.7) (21). The assay requires a coupled transcription/translation step to produce active enzyme molecules from the gene label, and this adds a degree of complexity to this assay format.

Many other nucleic acid labels have been proposed recently, including borane and borane hydride compounds, which are detected via reduction of a metal ion (e.g., silver) or a dye to a detectable product (22). A particularly intriguing direction in label technology is toward inorganic microparticle labels. These labels, named "nanocrystals" or "quantum dots" (23)(24), are very stable, water soluble, and can be as small as 2 nm in diameter. The particles have a shell-core construction. An outer shell of zinc sulfide surrounds a core containing a semiconductor material such as CdSe, InP, or InAs. Particle size and core composition determine the wavelength of emitted light, e.g., 2.1–4.6 nm diameter CdSe core particles give a blue emission, and 2.8–6.0 nm diameter InAs particles give an emission in the red region of the spectrum. The particles are rendered water soluble by either treatment with mercaptoacetic acid or the addition of a third outer layer of silica. The quantum dot labels have good quantum yields (30–50%) and good photochemical stability. Initial experiments with these labels were directed toward immunological applications (e.g., labeling F-actin filaments in fibroblast cells), and experiments with HeLa cells showed that the labels were biocompatible with living cells. These labels should also be useful in nucleic acid analyses, and such studies are underway.

multiple labels
Multiple labeling is another strategy to increase assay sensitivity. In the context of nucleic acid assays, avidin or streptavidin reagents provide one successful route to multiple labeling by virtue of the tetravalent interaction of these binding proteins with biotinylated conjugates (25). The branched DNA technology also introduces multiple labels onto a target nucleic acid and has achieved detection levels that rival other amplification methods (e.g., detection limit for HIV RNA is 50 molecules/mL). The sensitivity stems from the use of a set of branched reporter probes. Each probe has 15 branches, and each branch can react with up to three alkaline phosphatase-labeled detection probes. This leads to a high degree of labeling of the target (26). A newer route to multiple labeling exploits dendrimers. These are constructed from a set of complementary DNA probes that hybridize to form successive layers in a geometric expansion that produces a three-dimensional network onto which multiple labels can be attached (27). For example, a six-layer dendrimer has 2916 single-stranded arms for attachment of labeled probes. Results from Southern blot experiments have shown improvements of over 100-fold using labeled oligonucleotides plus dendrimers compared with labeled oligonucleotides alone (28).

background noise reduction
Improving the signal from a label is not the only way in which overall assay sensitivity can be improved. Background noise is often a more important limitation to ultrasensitivity. Current strategies for noise reduction include the use of time-resolved fluorescence labels that have relatively longer lifetimes of fluorescence compared with the short-lived background fluorescence. A delay between excitation and measurement of the fluorescence signal effectively removes any contribution from the background fluorescence. A recent microparticle assay for {Delta}F508 status based on this principle used 50-µm diameter microparticles coated with a capture DNA probe and europium chelate-labeled detection probes (17 europium atoms per probe) as reagents. The assay detected 1 x 106 target molecules per microparticle (signal-to-background ratio of 3), and this sensitivity was 100-fold lower than a comparable assay performed in a regular microwell format (29).

Immune complex transfer assays have been developed as a background reduction strategy for immunological reactions. The initial sandwich reaction occurs in the assay tube; the immune complex then is dissociated, transferred, and recaptured onto the walls of a separate antibody-coated detection tube. The net effect of the transfer is to leave nonspecifically bound interferents in the assay tube, thus eliminating their contribution to background as measured in the detection tube (30)(31)(32)(33)(34)(35). Application of this strategy to nucleic acid assays may also provide improvements in assay sensitivity.

Two more recent strategies that have been tested in nucleic acid assays include a photochemically activated cross-linking hybridization reaction to secure probes to complementary targets (36) and a multiphoton detection scheme for radioisotopic labels. The former has been validated in an assay for factor V Leiden status. Probes are labeled with a photoactive coumarin cross-linking agent. After hybridization, this label cross-links the specifically bound probes to the target, which can then be subjected to a higher stringency wash, thus eliminating nonspecifically bound materials more effectively. An additional benefit is that the covalent bond ensures that labeled probe is not dissociated and lost during subsequent pre-signal generation processing steps.

The multiphoton detection radioimmunoassay exploits the two-photon emission from 125I labels. Because the assay registers only two-photon emission events, the background falls from 20–70 counts per minute to 0.5 counts per week. As a result of this ultralow background, the detection limit for 125I is reduced to 0.1 zmol. This detection strategy was applied to a PCR assay (10–15 cycles) for the HIV-1 gag gene, and achieved a detection limit of 6 copies/mL (37).


   Convenient and Simple Assays
Top
 Abstract
 Introduction
 Labels and Sensitivity
 Convenient and Simple Assays
New Challenges for Nucleic...
 Conclusions
 References
 
There is still an unmet need for nucleic acid assays that are convenient for the analyst and simple to perform. In immunoassay, there is a wide choice of nonseparation (homogeneous) assay formats. The choice for nucleic acid assays is more limited. The LOCI assay is one example of a nonseparation assay format adapted to nucleic acid detection (38). It uses two populations of probe-coated particles: dye-loaded and signal-generating microparticles. Juxtaposition of the different types of microparticles, because of specific capture of target nucleic acid, facilitates effective transfer of singlet oxygen formed by photolysis at the surface of the dye-loaded particle to the signal-generating particle. This particle is loaded with an olefin that reacts with singlet oxygen to yield a chemiluminescent dioxetane compound that decomposes and sensitizes a fluorophore that re-emits the light.

Modulation of fluorescence polarization is the basis of a highly successful family of nonseparation immunoassays (39). This principle has only recently been applied to nucleic acid assays (40)(41)(42)(43). A fluorophore-labeled probe binds to target nucleic acid, and the polarization of the fluorescence of the bound label increases. This general assay principle had been applied to an amplification refractory mutation system-based test for {Delta}F508 status (44). Fluorescein was used as the label, and the assay detection limit was 10 nmol/L of amplicon. It has also been adapted to a strand displacement assays for Mycobacterium tuberculosis (5'-fluorescein and La Jolla Blue near infrared label) (45)(46)(47) and Chlamydia trachomatis (48), and PCR assays for methicillin-resistant Staphylococcus aureus (49) and Escherichia coli O157:H7 (50).

Simplicity and convenience are attributes required for assays designed for the point-of-care. Although the role of point-of-care nucleic acid testing is not well defined currently, several dipstick assay formats have been developed. These include assays for PCR products generated in an HIV-1 assay (51), K-ras mutations (52), and Salmonellae and Listeria spp in foods (53).

A rapid dipstick method for visual identification of specific DNA sequences has been developed based on DNA-tagged liposomes. The liposome particles (~0.2 µm diameter) were loaded with a red sulforhodamine B dye and tagged with a DNA probe. Target in the sample was hybridized to the DNA probe covalently linked to the liposome. This mixture was added to a tube containing a nitrocellulose dipstick with a zone of immobilized capture probe. Capillary migration transported the reaction mixture through the capture zone, which captured hybridized target: probe-liposome complexes to produce a visually discernible band. The assay could be completed in <10 min and detected 1 fmol of a synthetic 39mer target (54).


   New Challenges for Nucleic Acid Assays
Top
 Abstract
 Introduction
 Labels and Sensitivity
 Convenient and Simple Assays
 New Challenges for Nucleic...
Conclusions
 References
 
The advent of microminiaturization has presented new challenges for nucleic acid assays. Microchip and microarray devices have been developed as a result of the need for smaller analytical devices, e.g., for monitoring the release of biological warfare agents. They are also being developed to match the microminiaturization needs of the new massively parallel drug discovery strategies (55) and the need to screen for multiple mutations in genetic diseases (e.g., cystic fibrosis) (56) and multiple genes in expression assays (57)(58). The range of nucleic acid assays adapted to a microminiature format is extensive and includes different types of amplification reactions (e.g., PCR, reverse transcription-PCR, degenerate oligonucleotide primed-PCR, ligase chain reaction) (59)(60)(61)(62)(63)(64) and separation assays (capillary electrophoresis) (65). In addition, preanalytical steps such as cell isolation (66) and enzymatic digestion (e.g., in a restriction fragment length polymorphism assay) (67) have been miniaturized (Table 3 ). Fluorescence is the most common detection choice for chip-based nucleic acid assays, but chemiluminescence (68)(69)(70) and electrochemiluminescence (71) have also been adapted for microassay formats. Many microdevices are being constructed with internal volumes in the submicroliter range, and individual locations in a microdevice may have nanoliter or femtoliter volumes (72). Hence, the amount of substance to be detected in such small volumes is correspondingly small. This challenges even the most sensitive analytical methods.


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  		Table 3. Examples of microchip- and microarray-based nucleic acid assays.


   Conclusions
Top
 Abstract
 Introduction
 Labels and Sensitivity
 Convenient and Simple Assays
 New Challenges for Nucleic...
 Conclusions
References
 
There are many reasons for the quest for ultrasensitivity in nucleic acid analysis. These include analysis of genetic material from single cells and from rare cells, such as trophoblasts (73) in maternal circulation, and single-copy gene detection. In addition, better labels or detection reactions may provide a route to assays that require fewer amplification cycles or eliminate the multicycle protocols for nucleic acid amplification reactions (e.g., PCR). The multilabel branched DNA assay strategy and the emerging dendrimer-based assays indicate the potential for direct (nonamplification) assay formats. The fast pace of development of microchip and microarray devices poses new challenges for nucleic acid assays. Although at present fluorescence is the most widely used detection technique in microscale devices, many other detection technologies, such as those based on chemiluminescence, may play an important role in the future. This review has surveyed selected technologies; a glimpse of many other new labels, detection technologies, and formats for nucleic acids can be found in the recent World Wide Web-based full-text US patent database (74).


   Footnotes
 
Presented in part at the San Diego Conference, "DNA Technologies in Human Disease Detection", November 19–21, 1998, sponsored by the American Association for Clinical Chemistry and its San Diego Section, Molecular Pathology Division, and Lipid and Lipoprotein Division.


   References
Top
 Abstract
 Introduction
 Labels and Sensitivity
 Convenient and Simple Assays
 New Challenges for Nucleic...
 Conclusions
 References
 

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