| Recent advances make ‘Nucleic Acid Lateral Flow’ NALF a simple and functional platform that is a serious contender in the future NAT POC market. The nucleic acid test market has grown significantly in size and diversity over recent years. In general nucleic acid tests fit into three categories; those based on direct hybridisation and detection using specific nucleic acid probes; signal amplification, where specific probes bind the target sequence and the resulting signal is amplified; and target amplification, typically through enzymatic means. The third category is dominated by amplification methods that offer enhanced sensitivity, such as the polymerase chain reaction (PCR). Since its invention in the late 1980’s, the versatility of PCR has identified a multitude of commercial applications in the life sciences and in vitro diagnostics. More than 20 years later, PCR remains at the core of nucleic acid test technology and is common place in the clinical laboratory and essential as a research tool. Although PCR provides the benchmark for nucleic acid amplification, many alternative methods described in the literature have used novel and innovative approaches to achieve amplification and detection of target sequence. Such methodology includes nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), Qβ replicase, and simultaneous stand displacement amplification (SDA). The majority of these approaches translate to applications in infectious disease diagnostics, and although some have proven clinical utility, many may have been developed in an attempt to circumvent patent issues. More recently, research has concentrated on novel detection techniques and equipment designs that make PCR and other amplification technologies more amenable to modern day molecular diagnostic requirements. High-throughput automation of sample extraction, amplification and detection has dominated, primarily driven by blood bank screening for infectious diseases such as HIV, and of late by genomic screening and SNP analysis. Until recently point of care (POC) nucleic acid tests have remained elusive, largely due to the complexity of molecular assays and the technical challenges they present with regards to sample preparation and control of assay reproducibility and reliability. Nevertheless, fully-integrated POC nucleic acid platforms are emerging and several multinational companies from the diagnostics and IT industries have invested significant resource in the development of complex bioengineering strategies centred on microfluidic and bioelectronic sensor technologies. These devices are expensive and are currently limited to high-profile applications such as biodefense. An alternative and more cost effective approach to POC nucleic acid testing is to use a lateral flow platform. Lateral flow immunoassays are exemplified by pregnancy test devices and represent a significant proportion of today’s immunochemical POC market. The speed, low cost and simplicity of use make lateral flow immunoassays the only true point-of-care test. These chromatographic devices employ nanoparticles coated with materials which bind to an analyte, for example an antibody or antigen, within a sample. This analyte-nanoparticle complex flows laterally through a series of overlapping membranes and is captured on an antibody or antigen capture line. A visual result is achieved within a few minutes, allowing rapid interpretation by an unskilled operator without the need for complex or expensive equipment [Figure 1]. Nucleic acid lateral flow (NALF) uses nucleic acid hybridisation to capture and detect nucleic acid amplification products in a manner akin to lateral flow immunoassays, and this approach combines the advantages of such platforms with traditional nucleic acid tests. 
Figure 1. View of a LFI rapid test strip. NALF strips require modified test and capture lines and conjugate pad components. NALF Detection Strategies Capture Strategies Nucleic acids can be captured on lateral flow test strips in an antibody-dependent or antibody-independent manner. Antibody-dependent capture comprises an antibody capture line and a labelled amplicon or oligonucleotide probe of complementary sequence to the amplicon, e.g. goat anti-DNP and DNP-label [Figures 2A & B]. Antibody-independent alternatives offer more potential for multiplexing, minimise the potential for batch to batch variation and may reduce cost. One such method uses non-covalent interaction between two binding partners, for example the high affinity and irreversible linkage between a biotinylated probe or amplicon, and a streptavidin line [Figure 2C & D]. A much favoured and more simple approach is to immobilise oligonucleotide capture probes directly on to the nitrocellulose lateral flow membrane. This can be achieved by passive adsorption of a BSA-labelled oligonucleotide probe [Figure 2E], or more preferably an unlabelled oligonucleotide probe [Figure 2F]. All of these methods use standard lateral flow immunoassay striping equipment and yield strips with long term stability, often at room temperature. 
Figure 2. NALF capture strategies. A. Antibody stripe, labelled amplicon; B Antibody stripe, labelled oligonucleotide probe; C. Streptavidin stripe, biotinylated amplicon; D. Streptavidin stripe, biotinylated oligonucleotide probe; E. Passive adsorption of a BSA-oligonucleotide conjugate probe; F. Passive adsorption via non-covalent baking of an unlabelled oligonucleotide probe. Enzymatic Detection Signals In theory, any detection chemistry used in lateral flow immunoassays is also applicable to NALF, the limitation being the ability to conjugate the signal molecule of interest to an appropriate antibody or oligonucleotide detection probe. A broad range of NALF detection signals with quantitative and multiplexing capabilities is reported in the literature [Figure 3]. Reverse hybridisation enzymatic strip assays are the forerunner of today’s NALF tests. In these tests enzyme-labelled probes are hybridised to complementary nucleic acid target species on the surface of a nitrocellulose or nylon membrane resulting in a hapten-antibody-enzyme complex, for example biotin-streptavidin-alkaline phosphatase. Complex wash and substrate incubation steps are required to develop a readable signal. Therefore conversion to conventional ‘lateral’ flow suitable for non-laboratory nucleic acid test POC applications is relatively complex. For example, LFI-based Fluidics-on-FlexTM technology incorporates an integral fluidic circuit and pump manifold. 
Figure 3. NALF detection chemistries. In all cases the oligonucleotide probe may be replaced with an antibody and an appropriately labelled oligonucleotide probe or amplification primer. Enzyme, an enzyme-probe complex converts substrate into a colorimetric product; Gold, nanoparticles-probe conjugate visual signal; Qdot, Qdots-probe conjugate emits fluorescence; UPT, UPT-reporter-conjugate excited at 980nm and visible green light emitted at 500nm; Lipsome vesicle-probe conjugate releases dye. Nanoparticle Detection Signals Three ‘bead’ technologies have been successfully used in NALF, namely colloidal goId, latex and paramagnetic nanoparticles. Gold and latex both give rise to colorimetric signals visible to the naked eye or semi-quantifiable via inexpensive readers. Latex can be manufactured in any colour, whereas 2-250 nm gold nanoparticles have a characteristic red colour as a result of surface plasmon resonance [Figure 4]. Gold is the lateral flow nanoparticle of choice predominantly due to its small size, sensitivity, and robust manufacturing methods. It can be conjugated to antibodies and oligonucleotides, and labelled with small binding moieties such as biotin or DNP. Gold NALF platforms generally use 30-80nm nanoparticles conjugated to an anti-biotin antibody. This gold conjugate is then complexed with a biotinylated amplicon, or sequence-specific oligonucleotide detection probe which, when captured via an oligonucleotide capture probe or antibody-hapten based capture method, yields a signal in the form of line. The optimal gold nanoparticle size and concentration is dependent on assay specifications, including application, line intensity, colour (cherry red/purple), linear response to target concentration, and uniformity of multiplexed signals. 
Figure 4. Gold NALF detection labels appear red to the naked eye and comprise colloidal gold nanoparticle-antibody or -nucleic acid conjugates. Paramagnetic NALF uses 100-200 nm paramagnetic nanoparticles in a similar manner to gold NALF. These nanoparticles emit a non-visual signal when they are excited in a magnetic field and interpretation requires a specialised reader. Gold and superparamagnetic NALF can detect as little as 1 fmol of synthetic target, an order of magnitude greater than the sensitivity of labour-intensive gel electrophoresis. Superparamagnetic NALF promises further improvement. As for all nanoparticle detection technologies, quality reagents are a key pre-requisite; nanoparticles should be uniform in shape and size and remain aggregate free. A number of methods have been investigated to improve the sensitivity of nanoparticle NALF. Detection probes labelled with multiple hapten moieties have been used to form large signal enhancing lattices by specifically binding to multiple gold nanoparticle-conjugates. Combined with RT-PCR this method can achieve a visual sensitivity similar to that of fluorogenic instrument-based probe methods. Nanoparticle NALF may be combined with DNA dendrimer signal enhancement resulting in DNA dendrimers which are ‘branched’ nucleic acid species with 2-900 identical labels per dendrimer. They can improve biological assay sensitivities up to 200 fold, depending on dendrimer size, application and the nature of the assay. Methods are now available that allow gold and superparamagnetic particles to be coupled to oligonucleotide primers or probes. This approach may help minimise steric hindrance and maximise gold NALF assay sensitivity. By combining such approaches with oligonucleotide capture probe immobilisation, the need for antibody may also be eliminated. This antibody-free NALF format can reduce the number of assay components, and in some cases device cost, and with further optimisation this system may improve NALF sensitivity, specificity and reproducibility. It is also possible to prepare oligonucleotide gold and paramagnetic conjugates that are stable at elevated temperatures. Such conjugates can tolerate thermal PCR cycling conditions allowing them to be included in amplification reactions. Emerging Detection Chemistries More pioneering detection approaches draw on liposome and fluorescence methodology and are in early stages of development. Liposome nano-vesicles comprise a lipid bilayer that can be covalently linked to antibodies and oligonucleotides. These transparent spheres can be used to encapsulate aqueous signals such as dyes in a controlled manner. When employed in NALF, oligonucleotide tagged liposome nano-vesicles release dye to give a visual capture line, with a prototype device detecting as few as five viable Cryptosporidium oocysts. The use of fluorescence-based lateral flow immunoassay reporters is on the increase, and several have been demonstrated in NALF applications. For example, a dual fluorescein- and biotin-labelled oligo probe has been used to detect single stranded amplicon generated by cycling probe technology. Such standard fluorophores are limited by high background fluorescence, the need for a complex reader, and the number of spectrally diverse fluors available for multiplexing. UPT-NALF is an alternative approach which uses up-converting phosphor reporters; approximately 400 nm sized particles composed of rare earth lanthanide elements that are embedded in a crystal. These particles emit visible light after excitation with infrared in a process called up-conversion. Up-converting occurs in only the phosphor lattice, so auto-fluorescence of other assay components is virtually non-existent. UPT has been used to develop rapid pre-screening and hybridization-based confirmatory tests for the detection of human papillomavirus type 16, a marker for cervical cancers. A third fluorescence approach uses Quantum dots, or ‘Qdots’. These nanometer-sized semi-conductor nanocrystals have extraordinary optical fluorescence properties that can make them up to a thousand times brighter than conventional dyes. Nanocrystal size determines their colour, and emission is narrow and symmetric resulting in minimum cross-talk. Qdots are visualised under UV light and can be ‘tuned’ to allow excitation with the same long-wavelength UV lamp. The development of water-soluble Qdots that can be conjugated to antibodies (Qdot bioconjugates) has made Qdots amenable to LFI applications. Initial feasibility testing has used dot-infused hcG pregnancy tests and is likely to be extended to spectrally multiplexed assays and next generation NALF applications in the near future. Detection limits equivalent to or better than current gold-standard nucleic acid tests and lateral flow immunoassays are essential for many nucleic acid POC applications. All of these emerging technologies claim to improve on nanoparticle NALF and/or enzymatic detection signal sensitivity by 2-3 orders of magnitude, but are currently limited by the need for readers or early stage development. NALF Applications Low-Density Multiplexed NALF Automated high-throughput DNA chip and array technology is well suited to multiplexed detection of very high numbers of samples or probes. However, a need remains for low-density multiplexing platforms for POC detection in the region of 2-25 targets. Low-density multiplexed NALF fills this niche and is designed to detect multiplexed PCR amplicons such as TemplexTM assays. A simple multiplexed housed device comprises a single lateral flow strip, sample port and conjugate pad and multiple oligonucleotide capture probe stripes. A gold-conjugate based prototype device comprises seven different target and complementary probes and has been used to detect synthetic target mixes representing 14 different Influenza genotypes in 20 minutes at room temperature [Figures 5A & B]. Preliminary data demonstrates detection of as little as one femtomole of synthetic nucleic acid sequence, and one tenth of a standard TemplexTM reaction. Bi-directional housing that incorporates two longer test strips, or multi-directional housing, such as tri- and quad-NALF strips in a ‘T’ or cross-shape, can theoretically multiplex much higher numbers. In reality, the extent of multiplexing is limited by the availability of non-standard lateral flow membranes sizes, the rate and volume of flow through these, and the amount of gold-conjugate required. However, preliminary evidence suggests that 24-plex is feasible using a quad strip format. The major hurdle in multiplexed NALF is minimisation of non-specific signal inherent to NAT platforms comprising large numbers of probes and amplification primers, which can be achieved by careful optimisation of primer and probe sequences, concentrations, and capture line positions. 
Figure 5. Multiplexed NALF. A) Schematic describing conversion of ResPlexTM III: Influenza Typing Panel to Multiplexed NALF. B) 7plex detection of synthetic amplicon representing 14 different Influenza genotypes. 1, InfA N1 H1; 2, InfA, N1, H3; 3, InfA, N1,, H5; 4, InfA, N1 H undefined; 5, InfA, N2, H1; 6, InfA, N2, H3; 7, InfA, N2, H5; 8, InfA, N2, H undefined; 9, InfAB negative, H undefined; 10, InfA, H1, N undefined; 11, InfA, H3, N undefined; 12, InfA H5, N undefined; 13, InfA, HN undefined; 14, InfB, HN undefined. SNP NALF Single nucleotide polymorphisms (SNPs) are important indicators of human genetic disease, strain genotypes and drug resistance. A number of rapid SNP NALF detection techniques amenable to POC are in development. Most PCR-based SNP diagnostics use a single primer pair to make amplicons with variable internal regions containing one or more single base mismatches. One such NALF approach is competitive allele-specific short oligonucleotide hybridisation. This method discriminates at the capture level via biotinylated sequence-specific hybridisation probes designed to contain the SNP base of interest. Competition between labelled and unlabelled mutant and wild type probes results in presence or absence of signal at a streptavidin capture line in a target sequence-dependent manner [Figure 6A]. A disadvantage of this system is the requirement for separate reactions for each target sequence and post-amplification temperature ramping. However, an alternative approach uses allele-specific PCR and chimeric PCR primers that incorporate hexameric repeat tags termed ‘hexapet tags’. These tags are designed to have minimal cross-reactivity and have been used to demonstrate specific hybridisation-based capture of amplicon at room temperature using NALF strips striped with complementary hexapet tag sequences [Figure 6B]. This system discriminates between alleles at the amplification level with the disadvantage that multiple allele-specific primers are required. BBInternational is developing a non-competitive PCR NALF platform that discriminates at the detection level. Short immobilised hybrid nucleic acid probes are designed with carefully optimised melting temperature values [Figure 7A]. This system has demonstrated discrimination at room temperature without the need for allele-specific primers, and detects asymmetric PCR amplicon or amplicon that has been rendered single stranded by Lambda exonuclease enzyme digestion [Figure 7B]. All of these SNP-based NALF platforms can potentially be used to multiplex detection of 2 or more SNPs in a single device. In all cases the key to success is careful primer and/or probe design. 
Figure 6. SNP NALF detection strategies. A) Competitive allele-specific short oligonucleotide hybridisation (CASSOH). Biotinylated and unlabelled mutant and wild type sequence-specific hybridisation probes compete at the SNP site resulting in sequence-dependent presence or absence of signal at a streptavidin capture line. B) Hexapet tags. Chimeric PCR primers that incorporate hexameric repeat tags are used to synthesise PCR amplicons in an allele-specific manner. The resulting amplicons are captured at room temperature on complementary striped hexapet oligonucleotide probes. 
Figure 7. Bi-plex detection of SNPs using hybrid nucleic acid probes. A) Schematic. SNP-specific detection oligonucleotide probes are stripes capture single-stranded PCR amplicon. Anti-biotin-gold (red circle) conjugate and biotinylated (green circle) detection probe in dry conjugate pad format. B) Housed full-dipstick discrimination of Factor II wild type and mutant genotype sequences. C, wildtype; G, mutant. Generic & Immunochemical-NALF Combination Formats Methods are available that allow manufacture of generic NALF strips. Such strips comprise a bridge oligo with a first region complementary to a generic striped oligonucleotide probe and a second sequence-specific region complementary to the amplicon of interest [Figure 8A]. Detection of different target sequences requires re-design of the bridge oligo but uses the same generic NALF strip, theoretically minimising strip redesign. An extension of this generic approach is the development of combination lateral flow immunoassay strips that utilise nucleic acid base pairing to facilitate detection of immunochemical targets [Figure 8B]. Detection involves a generic striped oligonucleotide probe and a complementary oligonucleotide-human antibody conjugate. The analyte of interest is ‘sandwiched’ between the capture conjugate and a cross-reacting antigen-label conjugate. 
Figure 8. Generic NALF. A) Bridging Probe NALF. Facilitated by a bridging oligonucleotide probe with a first portion complementary to a labelled PCR product and a second portion complementary to a striped capture oligonucleotide. B) Immuno-NALF combinatorial sandwich assay approach to detect patient antibody. Patient antibody (red) is sandwiched between an antigen-labelled conjugate detection reagent and an anti-human antibody-oligonucleotide conjugate. The latter facilitates capture of the labelled complex via a complementary striped capture oligonucleotide. Design Criteria, Manufacturing Implications and Limitations The majority of thermal and isothermal nucleic acid test amplification technologies can be readily converted to NALF by adaptation of probes and labelling moieties. In many cases amplification and detection probe sequence re-design is unnecessary, resulting in minimum development time and cost, and potentially reduced timescales for regulatory approval. The incorporation of amplification and detection controls is also relatively straight forward by inclusion of one or more additional capture lines and complementary probes and/or primers. When designing NALF assays, general lateral flow immunoassay design criteria that influence assay sensitivity and non-specific signal must be taken into account. Nucleic-acid compatible sample and conjugate pads and blocking materials must be chosen. For example, nucleic acids will ‘stick’ to glass-fibre materials causing sample retention and minimising probe release, however these effects can be reduced through the application of novel pad materials and blocking strategies. Nitrocellulose membrane pore size and flow rate are also key as there is a trade-off between assay time and efficient target-probe hybridisation. NALF detection is not particularly prone to sample effects as the sample is often diluted in upstream processes, for example amplification steps. Gold NALF can detect amplification reactions that contain blood products and crude bacterial cell lysates without significant signal inhibition so that, unlike lateral flow immunoassays, complex sample pad materials for upstream processes such as blood separation are not always necessary. Future NALF devices are likely to include dry format amplification and detection reagent technology and versatile lateral flow housing designs. Antibody and oligonucleotide-nanoparticle conjugates can already be supplied in dry format, soaked conjugate pad or sprayed reagent line. Technology is also available to combine PCR and nanoparticle NALF components as a dry pellet that can be reconstituted upon sample addition. NALF strip manufacture uses standard chromatographic lateral flow materials, and strong similarity between NALF probe and antibody immobilisation methodologies allows the use of standard automated lateral flow manufacturing equipment, minimising equipment costs and eliminating the need to re-train production staff. Unmodified oligonucleotides are significantly cheaper than striped antibodies, and oligonucleotide striped membranes have long term stability at room temperature. Successful NALF detection chemistries will be operated and stored at room temperature. Modern day lateral flow immunoassay designs can be extremely elaborate, incorporating novel functions such as separate sample and buffer ports, buffer bags and blister packs that entail piercing or wick insertion, multi-directional flow, multiple analyte detection, and moving parts [Figure 9]. Standard injection moulded housings often hide complex mechanisms such as wash steps behind simple push button fascias. Similar approaches are being used to develop novel ‘closed tube’ NALF housing designs that meet future POC diagnostic needs in the clinical, environmental and food diagnostic sectors. For example, BBInternational is developing housing that allows cross-contamination-free transfer of a PCR reaction to a NALF test strip. 
Figure 9. A range of diverse lateral flow housing designs. NALF detection is rapid and has been achieved in as little as five minutes, dependent on application and detection scheme. A major future challenge is the incorporation of rapid, simple and relatively inexpensive upstream sample extraction and amplification processes that reduce the overall time-to-result. Future NALF devices are likely to incorporate emerging technologies that reduce these bottle-neck processes to a matter of minutes. For example, the Rapid Cycler 2 achieves amplification in 15 minutes but comes with a high price tag. Future microfluidic PCR devices may prove simpler, less expensive, and more amenable to NALF. Prototypes are emerging that have no moving parts or complex in situ components such as pumps, valves, or wells, and perform PCR in less than five minutes with the same yield as a typical 90 minute thermal cycler protocol. Similarly rapid and expensive bench-top extraction equipment is also emerging. For example, the PlasmaGen APR-510-S provides rapid, one-step extraction of DNA or RNA in 1-2 min from a variety of dry sample matrices, and the QuickGene-810 isolates DNA and RNA from whole blood in six minutes. Another approach uses rapid cycles of hydrostatic pressure to extract biological material. All of these methods yield high quality nucleic acid from low abundant samples and are potentially NALF-compatible, but there remains a need for low cost, simple alternatives. A variety of manufacturers offer magnetic bead systems that avoid centrifugation but these generally require ‘open-tube’ transfer from lysis tube to amplification vessel. Some progress has been made with the development of chaotropic archiving materials. These ‘papers’ contain chemicals that lyse cells, denature proteins and protect nucleic acids from nucleases, oxidation and UV damage. Unfortunately, unfavourable wash steps are also necessary. Tubes coated with a solid-phase matrix that irreversibly binds nucleic acid, allowing extraction and PCR in the same reaction vessel, are another step in the right direction. Future solutions may also lie in improved amplification technologies that can tolerate cruder sample extracts. Elements of the described NALF technologies draw on skills ‘known in the art’, whilst others are patent-protected or based upon carefully guarded know-how. When choosing a NALF developer it is important to also consider the impact of early immunochromatographic lateral flow patents. BBInternational has negotiated a unique agreement with Inverness Medical Innovations that offers protection for contract development and manufacturing customers on a selection of lateral flow patents within the Inverness portfolio. Conclusions Recent technological advances in nanoparticle and alternative detection chemistries, and the development of flexible platforms that allow multiplexing and SNP detection make NALF a serious contender in the future nucleic acid POC market. This rapid, simple and inexpensive detection methodology has potential applications in rapid infectious disease strain typing, human genetic disease diagnosis, and environmental field-site testing. Development of sensitive and accurate NALF POC tests will most likely be achieved through interdisciplinary partnering between molecular biology specialists, experienced immunochemical lateral-flow developers and manufacturers, and experts in sample extraction, amplification processes and equipment design. This approach will allow full exploitation of this flexible technology, taking a step closer to true POC nucleic acid tests. |