Nicholas M. Adams

Development of a low-resource RNA extraction cassette based on surface tension valves.

Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low-resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low-resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid-air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low-resource setting.

Design, synthesis, and characterization of nucleic-acid-functionalized gold surfaces for biomarker detection.

Nucleic-acid-functionalized gold surfaces have been used extensively for the development of biological sensors. The development of an effective biomarker detection assay requires careful design, synthesis, and characterization of probe components. In this Feature Article, we describe fundamental probe development constraints and provide a critical appraisal of the current methodologies and applications in the field. We discuss critical issues and obstacles that impede the sensitivity and reliability of the sensors to underscore the challenges that must be met to advance the field of biomarker detection.

Design criteria for developing low-resource magnetic bead assays using surface tension valves

Many assays for biological sample processing and diagnostics are not suitable for use in settings that lack laboratory resources. We have recently described a simple, self-contained format based on magnetic beads for extracting infectious disease biomarkers from complex biological samples, which significantly reduces the time, expertise, and infrastructure required. This self-contained format has the potential to facilitate the application of other laboratory-based sample processing assays in low-resource settings. The technology is enabled by immiscible fluid barriers, or surface tension valves, which stably separate adjacent processing solutions within millimeter-diameter tubing and simultaneously permit the transit of magnetic beads across the interfaces. In this report, we identify the physical parameters of the materials that maximize fluid stability and bead transport and minimize solution carryover. We found that fluid stability is maximized with ≤0.8 mm i.d. tubing, valve fluids of similar density to the adjacent solutions, and tubing with ≤20 dyn/cm surface energy. Maximizing bead transport was achieved using ≥2.4 mm i.d. tubing, mineral oil valve fluid, and a mass of 1-3 mg beads. The amount of solution carryover across a surface tension valve was minimized using ≤0.2 mg of beads, tubing with ≤20 dyn/cm surface energy, and air separators. The most favorable parameter space for valve stability and bead transport was identified by combining our experimental results into a single plot using two dimensionless numbers. A strategy is presented for developing additional self-contained assays based on magnetic beads and surface tension valves for low-resource diagnostic applications.

Comparison of three magnetic bead surface functionalities for RNA extraction and detection.

Magnetic beads are convenient for extracting nucleic acid biomarkers from biological samples prior to molecular detection. These beads are available with a variety of surface functionalities designed to capture particular subsets of RNA. We hypothesized that bead surface functionality affects binding kinetics, processing simplicity, and compatibility with molecular detection strategies. In this report, three magnetic bead surface chemistries designed to bind nucleic acids, silica, oligo (dT), and a specific oligonucleotide sequence were evaluated. Commercially available silica-coated and oligo (dT) beads, as well as beads functionalized with oligonucleotides complementary to respiratory syncytial virus (RSV) nucleocapsid gene, respectively recovered ∼75, ∼71, and ∼7% target RSV mRNA after a 1 min of incubation time in a surrogate patient sample spiked with the target. RSV-specific beads required much longer incubation times to recover amounts of the target comparable to the other beads (∼77% at 180 min). As expected, silica-coated beads extracted total RNA, oligo (dT) beads selectively extracted total mRNA, and RSV-specific beads selectively extracted RSV N gene mRNA. The choice of bead functionality is generally dependent on the target detection strategy. The silica-coated beads are most suitable for applications that require nucleic acids other than mRNA, especially with detection strategies that are tolerant of a high concentration of nontarget background nucleic acids, such as RT-PCR. On the other hand, oligo (dT) beads are best-suited for mRNA targets, as they bind biomarkers rapidly, have relatively high recovery, and enable detection strategies to be performed directly on the bead surface. Sequence-specific beads may be best for applications that are not tolerant of a high concentration of nontarget nucleic acids that require short RNA sequences without poly(A) tails, such as microRNAs, or that perform RNA detection directly on the bead surface.

Automated Device for Asynchronous Extraction of RNA, DNA, or Protein Biomarkers from Surrogate Patient Samples

Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein).

The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry

Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼105 target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

A handheld orbital mixer for processing viscous samples in low resource settings

Clinical diagnostics play an important role in infectious disease monitoring. Often these diseases are endemic in settings that lack laboratory resources, which limits the types of tools that are available. Mixing is a common procedure in clinical diagnostic assays and is not immune to these challenges. In this report, we characterize the efficacy of a handheld therapeutic massager that has been repurposed into a mixer. Using metal affinity functionalized magnetic beads and a histidine-tagged fluorescent label, we find that this inexpensive orbital mixer is effective over a range of clinically relevant sample volumes and viscosities. Even with larger sample volumes (>200 μL) and more viscous sample matrices (3× ηH2O), the mixer binds more than 90% of the biomarkers in solution in less than 90 seconds. In a clinical assay for the detection of Plasmodium falciparum histidine-rich protein 2, we find there is no statistical difference in performance between our inexpensive and portable orbital mixer and a standard laboratory benchtop vortexer.

Adaptive PCR Based on Hybridization Sensing of Mirror-Image l-DNA

Polymerase chain reaction (PCR) is dependent on two key hybridization events during each cycle of amplification, primer annealing and product melting. To ensure that these hybridization events occur, current PCR approaches rely on temperature set points and reaction contents that are optimized and maintained using rigid thermal cycling programs and stringent sample preparation procedures. This report describes a fundamentally simpler and more robust PCR design that dynamically controls thermal cycling by more directly monitoring the two key hybridization events during the reaction. This is achieved by optically sensing the annealing and melting of mirror-image l-DNA analogs of the reactions primers and targets. Because the properties of l-DNA enantiomers parallel those of natural d-DNAs, the l-DNA reagents indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reaction. This hybridization-sensing approach adapts in real time to variations in reaction contents and conditions that impact primer annealing and product melting and eliminates the requirement for thermal calibrations and cycling programs. Adaptive PCR is demonstrated to amplify DNA targets with high efficiency and specificity under both controlled conditions and conditions that are known to cause traditional PCR to fail. The advantages of this approach promise to make PCR-based nucleic acid analysis simpler, more robust, and more accessible outside of well-controlled laboratory settings.

Rapid concentration and elution of malarial antigen histidine-rich protein II using solid phase Zn(II) resin in a simple flow-through pipette tip format

Rapid diagnostic tests (RDTs) designed to function at the point of care are becoming more prevalent in malaria diagnostics because of their low cost and simplicity. While many of these tests function effectively with high parasite density samples, their poor sensitivity can often lead to misdiagnosis when parasitemia falls below 100 parasites/μl. In this study, a flow-through pipette-based column was explored as a cost-effective means to capture and elute more Plasmodium falciparum histidine-rich protein II (HRPII) antigen, concentrating the biomarker available in large-volume lysed whole blood samples into volumes compatible with Plasmodium falciparum-specific RDTs. A systematic investigation of immobilized metal affinity chromatography divalent metal species and solid phase supports established the optimal design parameters necessary to create a flow-through column incorporated into a standard pipette tip. The bidirectional flow inherent to this format maximizes mixing efficiency so that in less than 5 min of sample processing, the test band signal intensity was increased up to a factor of twelve from HRPII concentrations as low as 25 pM. In addition, the limit of detection per sample was decreased by a factor of five when compared to the RDT manufacturers suggested protocol. Both the development process and commercial viability of this application are explored, serving as a potential model for future applications.