Sensitive Nucleic Acid Detection Research Articles

Addressing the Analytic Challenges of Cross-Sectional Pediatric Pneumonia Etiology Data.

Despite tremendous advances in diagnostic laboratory technology, identifying the pathogen(s) causing pneumonia remains challenging because the infected lung tissue cannot usually be sampled for testing. Consequently, to obtain information about pneumonia etiology, clinicians and researchers test specimens distant to the site of infection. These tests may lack sensitivity (eg, blood culture, which is only positive in a small proportion of children with pneumonia) and/or specificity (eg, detection of pathogens in upper respiratory tract specimens, which may indicate asymptomatic carriage or a less severe syndrome, such as upper respiratory infection). While highly sensitive nucleic acid detection methods and testing of multiple specimens improve sensitivity, multiple pathogens are often detected and this adds complexity to the interpretation as the etiologic significance of results may be unclear (ie, the pneumonia may be caused by none, one, some, or all of the pathogens detected). Some of these challenges can be addressed by adjusting positivity rates to account for poor sensitivity or incorporating test results from controls without pneumonia to account for poor specificity. However, no classical analytic methods can account for measurement error (ie, sensitivity and specificity) for multiple specimen types and integrate the results of measurements for multiple pathogens to produce an accurate understanding of etiology. We describe the major analytic challenges in determining pneumonia etiology and review how the common analytical approaches (eg, descriptive, case-control, attributable fraction, latent class analysis) address some but not all challenges. We demonstrate how these limitations necessitate a new, integrated analytical approach to pneumonia etiology data.

Nucleic acid detection with CRISPR-Cas13a/C2c2.

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.

Polymer Brush‐Modified Microring Resonators for Partition‐Enhanced Small Molecule Chemical Detection

AbstractSilicon photonic microring resonators have emerged as a promising technology for the sensitive detection of biological macromolecules, including proteins and nucleic acids. However, not all species of interest are large biologics that can be targeted by highly specific capture agents. For smaller organic chemicals, including many toxic and regulated species, a general approach to improving sensitivity would be desirable. By functionalizing the surface of silicon photonic microring resonators with polymer brushes, small molecules can selectively partition into the surface‐confined sensing region of the optical resonators. This in turn leads to response enhancements in excess of 1000% percent, relative to non‐functionalized sensors, for representative targets including 4‐methylumbelliferyl phosphate, a simulant for highly toxic organophosphates, Bisphenol A, an industrial pollutant, as well as other small organic analytes of interest. There are many polymer brush chemistries compatible with silicon resonators, making this a general strategy towards tuning sensor selectivity and specificity by optimizing interactions between the agent(s) of interest and the polymer construct.

Paper microchip with a graphene-modified silver nano-composite electrode for electrical sensing of microbial pathogens.

Rapid and sensitive point-of-care diagnostics are of paramount importance for early detection of infectious diseases and timely initiation of treatment. Here, we present cellulose paper and flexible plastic chips with printed graphene-modified silver electrodes as universal point-of-care diagnostic tools for the rapid and sensitive detection of microbial pathogens or nucleic acids through utilizing electrical sensing modality and loop-mediated isothermal amplification (LAMP). We evaluated the ability of the developed paper-based assay to detect (i) viruses on cellulose-based paper microchips without implementing amplification in samples with viral loads between 106 and 108 copies per ml, and (ii) amplified HIV-1 nucleic acids in samples with viral loads between 10 fg μl-1 and 108 fg μl-1. The target HIV-1 nucleic acid was amplified using the RT-LAMP technique and detected through the electrical sensing of LAMP amplicons for a broad range of RNA concentrations between 10 fg μl-1 and 108 fg μl-1 after 40 min of amplification time. Our assay may be used for antiretroviral therapy monitoring where it meets the sensitivity requirement of the World Health Organization guidelines. Such a paper microchip assay without the amplification step may also be considered as a simple and inexpensive approach for acute HIV detection where maximum viral replication occurs.

Ultrasensitive paper based nucleic acid detection realized by three-dimensional DNA-AuNPs network amplification

A novel three-dimensional DNA-AuNPs network structure amplification strategy was employed to design a lateral flow biosensor by introducing streptavidin coated gold nanoparticles (Au-SA) in this paper. They act as amplification probes which aggregate numerous gold nanoparticles (AuNPs) on test line by forming a three-dimensional DNA-AuNPs network structure in the presence of target. Sensitive detection of nucleic acid with point-of-care analysis is significant for infectious agent, early diagnosis and treatment of genetic diseases. The use of these particles in rapid ultrasensitive point of care (POC) lateral flow assays lead to a linear range from 0.1pM to 250nM with a limit of detection of 0.01 pM without polymerase chain reaction (PCR). The proposed method could increase the sensitivity by 4 orders of magnitudes than traditional sandwich assays labeled with AuNPs. Furthermore, the assay owns good reproducibility and stability, which will prove practical diagnostic applications.

PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor.

A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100–1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.

Superhydrophilic cotton thread with temperature-dependent pattern for sensitive nucleic acid detection

Cotton thread is promising in fabricating biosensors for diagnostic application due to its excellent characteristics. However, the enrichment of the capture molecules on a narrow zone of the cotton thread based biosensor is a big challenge because of its superhydrophilicity. Here, we report a simple, low-cost and accurate cotton thread based nucleic acid biosensor with temperature-dependent pattern. Liquid wax is used to fabricate temperature-dependent pattern to restrict the test zone in a narrow area. This biosensor enables visual and quantitative detection of target DNA by accumulation of gold nanoparticles (GNPs) on the test zone with a detection limits of 0.75nM. In addition, the cotton thread based biosensor needs less sample than previous reported lateral flow strip and the sample solution wicks faster at the cotton thread which can lead to a shorter detection time. This simple, low-cost and fast detection method holds great potential to improve healthcare services in the developing regions.

Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles

Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10−18 mol l−1 for t-DNA has been achieved.

Biofouling-Resilient Nanoporous Gold Electrochemical Sensors for DNA Detection

Introduction: Rapid detection of biomolecular markers is essential for early diagnosis and management of diseases as well as for fundamental biological studies. Electrochemical sensors have been powerful tools for addressing this need, where nanostructured electrodes have shown significant promise in enhancing the performance of these sensors. In order to develop electrochemical biosensors with rationally-designed nanostructures, it is imperative to have a fundamental understanding of the structure-property relationships that dictate sensor performance. In this study, microfabrication-compatible nanoporous gold (np-Au) thin films with tunable pore morphology are used as a model electrode to investigate the effect of electrode morphology on sensor performance.Specifically, by modifying np-Au pore morphology (i.e., pore size, effective surface area) and employing electrochemical characterization, we studied the effect of morphology on (i) grafting density of 26mer DNA probes, (ii) limit of detection in hybridization to a DNA target, and (iii) dynamic range in detecting different target concentrations1. We subsequently assessed the performance of np-Au electrodes for nucleic acid detection in complex biological media, with the hypothesis that the optimal pore geometry should allow permeation of nucleic acids into the porous electrode while sieving out biofouling molecules. Results and Discussion: Np-Au samples were fabricated by sputtering gold/silver alloy on glass coverslips and subsequent etching of silver to self-assemble the np-Au films (un-annealed np-Au). To obtain samples with different pore morphology (annealed np-Au), the films were thermally-treated at 225 °C. The np-Au sensor displayed a 100-fold shift in dynamic range of detection towards lower concentrations of DNA (10 nm – 200 nM) as compared to a planar gold electrode. Annealed np-Au films showed improved accessibility and a further 10-fold shift in dynamic range (Figure 1). The hybridization on np-Au films can be viewed as a combination of two events: (i) target DNA entering the pores and (ii) target DNA binding with probe upon entering the pore. Geometric accessibility determines the efficiency of a target entering the porous structure. Once inside the porous structure, the probability of a target binding to the probe depends on probe density. Target molecule encounters a significantly higher number of probes within a pore compared to a planar surface. These two factors together (i.e., enhanced accessibility and higher probe grafting density in porous films) led to the detection of lower concentrations of target and resulted in a shift in dynamic range of detection with morphology (Figure 1). A major obstacle to nucleic acid detection in biological fluids is electrode fouling by constituents that obscure the transport of analytes to the active electrode surfaces. Np-Au electrodes circumvent this by enabling DNA detection in physiologically relevant complex media (bovine serum albumin and fetal bovine serum (FBS))2. In contrast, sensor performance was compromised for planar gold electrodes in the same conditions. Un-annealed np-Au electrodes produced a signal suppression (hybridization efficiency) of ~38% with a dynamic range of 25 nM to 200 nM (Figure 2) in FBS. Hybridization efficiency decreased by 10% for np-Au with coarser pores (annealed np-Au) revealing a pore size dependence of sensor performance in biofouling conditions. This nanostructure dependent functionality in complex media suggests that pores with the optimal size act as sieves for blocking the biomolecules from inhibiting the surfaces within the porous volume while allowing the transport of nucleic acids analytes and redox molecules Conclusions: In summary,we demonstrated that np-Au electrodes exhibit high electrochemical sensing performance in the presence of common biofouling media and allow for sensitive electrochemical detection of nucleic acids. The un-annealed np-Au electrodes exhibited the highest signal suppression followed by annealed np-Au and planar Au electrodes in presence of FBS. This indicates that pore size plays a significant role in dictating the extent of biomolecule adsorption and transport. This novel technology for detecting target DNA concentrations in tunable dynamic ranges, coupled with sustained functionality in complex biological media, should assist in the development of robust point-of-care diagnostic platforms.

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

AbstractA new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP‐releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with Km values averaging 13‐fold higher than those of natural dNTPs, and kcat values within 1.5‐fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

ATP-Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling.

A new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP-releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with Km values averaging 13-fold higher than those of natural dNTPs, and kcat values within 1.5-fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

Self-assembled biosensor with universal reporter and dual-quenchers for detection of unlabelled nucleic acids.

A novel biosensor with universal reporter and dual quenchers was developed for rapid, sensitive, selective, and inexpensive detection of unlabelled nucleic acids. The biosensor is based on a single-strand DNA stem-loop motif with an extended universal reporter-binding region, a G-base rich stem region, and a universal address-binding region. The self-assembly of these stem-loop probes with fluorescence labeled universal reporter and a universal address region conjugated to gold nanoparticles forms the basis of a biosensor for DNA or microRNA targets in solution. The introduction of dual quenchers (G-base quenching and gold surface plasmon resonance-induced quenching) significantly reduces the fluorescence background to as low as 12% of its original fluorescence intensity and hence enhances the detection limit to 0.01 picomoles without signal ampilication.

High sensitive and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles.

A novel class of probes for fluorescence detection was developed and combined to microgel particles for a high sensitive fluorescence detection of nucleic acids. A double strand probe with an optimized fluorescent-quencher couple was designed for the detection of different lengths of nucleic acids (39 nt and 100 nt). Such probe proved efficient in target detection in different contests and specific even in presence of serum proteins. The conjugation of double strand probes onto polymeric microgels allows for a sensitive detection of DNA sequences from HIV, HCV and SARS corona viruses with a LOD of 1.4 fM, 3.7 fM and 1.4 fM, respectively, and with a dynamic range of 10(-9)-10(-15) M. Such combination enhances the sensitivity of the detection of almost five orders of magnitude when compared to the only probe. The proposed platform based on the integration of innovative double strand probe into microgels particles represents an attractive alternative to conventional sensitive DNA detection technologies that rely on amplifications methods.

Au nanoparticles fluorescence switch-mediated target recycling amplification strategy for sensitive nucleic acid detection

A sensitive strategy based on Au nanoparticles fluorescence switch-mediated target recycling amplification for nucleic acid detection.

Molecular Detection of Schistosome Infections with a Disposable Microfluidic Cassette

Parasitic helminths such as schistosomes, as well as filarial and soil-transmitted nematodes, are estimated to infect at least a billion people worldwide, with devastating impacts on human health and economic development. Diagnosis and monitoring of infection dynamics and efficacy of treatment depend almost entirely on methods that are inaccurate, labor-intensive, and unreliable. These shortcomings are amplified and take on added significance in mass drug administration programs, where measures of effectiveness depend on accurate monitoring of treatment success (or failure), changes in disease transmission rates, and emergence of possible drug resistance. Here, we adapt isothermal molecular assays such as loop-mediated isothermal amplification (LAMP) to a simple, hand-held, custom-made field-ready microfluidic device that allows sensitive and specific detection of schistosome cell-free nucleic acids in serum and plasma (separated with a point-of-care plasma separator) from Schistosoma mansoni-infected mice. Cell-free S. mansoni DNA was detected with our device without prior extraction from blood. Our chip exhibits high sensitivity (~2x10−17 g/μL), with a positive signal for S. mansoni DNA detectable as early as one week post infection, several weeks before parasite egg production commences. These results indicate that incorporation of isothermal amplification strategies with our chips could represent a strategy for rapid, simple, low-cost diagnosis of both pre-patent and chronic schistosome infections as well as potential monitoring of treatment efficacy.

Ultraspecific electrochemical DNA biosensor by coupling spontaneous cascade DNA branch migration and dual-signaling sensing strategy

Using spontaneous cascade DNA branch migration and dual-signaling sensing strategy, we developed a novel universal electrochemical biosensor for the highly specific and sensitive detection of nucleic acids. A target strand (Ts) competitively hybridized with a ferrocene (Fc)-labeled signal probe (Fc-S1), which was blocked by a protector strand (Ps), after strand displacement to form the Ts/Fc-S1 duplex. A methylene blue (MB)-modified signal probe (MB-S2) was immobilized on the Au electrode surface by hybridizing with a thiolated capture probe (Cp). Then, the obtained reactants (Ts/Fc-S1 and MB-S2/Cp) suffered spontaneous DNA branch migration and produced two hybridization products (Fc-S1/Cp and MB-S2/Ts). These reactions led to the dissociation of MB molecules and the collection of Fc molecules. The detection mechanism of this DNA biosensor involved distance variation between the redox tags and the Au electrode, which was associated with target-induced cascade DNA branch migration. Moreover, we rationally designed the cascade DNA branch migration to occur spontaneously with ΔG°≈0, at which slight thermodynamic changes caused by base mismatch exerted a disproportionately large effect on the hybridization yield. This “signal-on/off” sensing system exhibited a remarkable analytical performance and an ultrahigh discrimination capability even against a single-base mismatch. The maximum discrimination factor (DF) of base mutations or alterations can reach 17.9. Therefore, our electrochemical biosensor might hold a great potential for further applications in biomedical research and early clinical diagnosis.

Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins

Sensitive and specific detection of nucleic acids and proteins plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids and proteins (thrombin) at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform.

Application Progress of Exonuclease-Assisted Signal Amplification Strategies in Biochemical Analysis

Abstract As an important member of the tool enzymes, exonuclease is a kind of hydrolytic enzymes without strict base sequence dependence. In recent years, by taking advantage of different hydrolysis ways of exonuclease and nanotechnology, cycle effect of enzyme digestion, aptamer, non Watson-Crick base pairing system by metal ions, fluorescent nucleic acid probes, electrochemical methods etc., a series of exonuclease-assisted signal amplification strategies have been developed, which have played a key role in improving the sensitivity of detection method. Therefore, exonucleases have been widely used in highly sensitive detection of nucleic acids, proteins, ions, small molecules and so on. To understand it better and apply it well in the future, the application progresses of exonuclease-assisted signal amplification strategies in biochemical analysis have been summarized in this review.

Tracing Phosphate Ions Generated during Loop-Mediated Isothermal Amplification for Electrochemical Detection of Nosema bombycis Genomic DNA PTP1.

Traditionally, amplified DNA detection in a loop-mediated isothermal amplification (LAMP) was carried out in a complicated gel electrophoresis or with expensive fluorescence-based methods. Here, instead of direct detection that relies on amplified DNA, the indirect detection based on tracing phosphate ions (Pi) generated during LAMP by using an electrochemical method has been proposed for sensitive nucleic acid detection. Pyrophosphate (PPi) as the byproduct of nucleic acid polymerization reaction in LAMP was hydrolyzed into Pi by the preaddition of thermostable inorganic pyrophosphatase (PPase). Thus, the total amount of Pi in the LAMP-amplified sample was proportional to the amount of starting DNA templates. The obtained Pi could then react with acidic molybdate to form the molybdophosphate precipitates on the electrode surface, which serve as redox mediators to give a readily measurable electrochemical signal. The practicality of this strategy has been further demonstrated by employing it for sensitive and accurate quantification of Nosema bombycis genomic DNA PTP1. The electrochemical method allowed the quantitative analysis for target genomic DNA with a detection limit of 17 fg/μL. Thus, we suppose that the novel method proposed in this work with superior sensitivity and specificity, as well as the simple feature, can be easily established for quantitative analysis of many other kinds of nucleic acids in the assistance of LAMP.

Ultratrace DNA Detection Based on the Condensing-Enrichment Effect of Superwettable Microchips.

A sensitive nucleic acid detection platform based on superhydrophilic microwells spotted on a superhydrophobic substrate is fabricated. Due to the wettability differences, ultratrace DNA molecules are enriched and the fluorescent signals are amplified to allow more sensitive detection. The biosensing interface based on superwettable materials provides a simple and cost-effective way for ultratrace DNA sensing.