Lateral Flow Nucleic Acid Biosensor Research Articles

Visual detection of microRNAs using gold nanorod-based lateral flow nucleic acid biosensor and exonuclease III-assisted signal amplification.

An ultrasensitive method for the visual detection of microRNAs (miRNAs) in cell lysates using a gold nanorod-based lateral flow nucleic acid biosensor (GN-LFNAB) and exonuclease III (Exo III)-assisted signal amplification. The Exo III-catalyzed target recycling strategy is employed to generate a large number of single-strand DNA products, which can be detected by GN-LFNAB visually. With the implementation of a unique recycling strategy, we have demonstrated that the miRNA in the concentration as low as 0.5pM can be detected without the need for instrumentation, providing a detection limit that surpasses previous reports. The new biosensor is ultrasensitive and can be applied to the reliable monitoring of miRNAs in cell lysates with high accuracy. The approach offers a simple and rapid tool for cancer diagnosis and clinical biomedicine, thanks to its flexibility, simplicity, cost-effectiveness, and convenience. This new method has the potential to significantly improve the detection and monitoring of cancer biomarkers, ultimately contributing to more effective cancer diagnosis and treatment.

AuPt nanoalloy with dual functionalities for sensitive detection of HPV16 DNA.

Human papillomavirus type 16 (HPV16), one of the high-risk types, is responsible for 53% of cervical cancers. The development of an early diagnostic approach with high sensitivity, low-cost, point-of-care testing (POCT) for HPV16 is urgent. In our work, a novel dual-functional AuPt nanoalloy-based lateral flow nucleic acid biosensor (AuPt nanoalloy-based LFNAB) was established with excellent sensitivity for detecting HPV16 DNA for the first time. The AuPt nanoalloy particles were prepared by a one-step reduction method, which was simple, rapid, and green. The AuPt nanoalloy particles retained the performance of initial Au nanoparticles owing to the catalytic activity enabled by Pt. Such dual-functionalities offered two kinds of detection alternatives (i.e., normal mode and amplification mode, respectively). The former is produced just by the black color from the AuPt nanoalloy material itself, and the latter is more color sensitive from its superior catalytic activity. The optimized AuPt nanoalloy-based LFNAB exhibited satisfactory quantitative ability in detecting the target HPV16 DNA in the range of 5-200 pM with a LOD of 0.8 pM at the "amplification mode". The proposed dual-functional AuPt nanoalloy-based LFNAB displayed great potential and promising opportunity in POCT clinical diagnostics.

Paper-based detection of Epstein-Barr virus using asymmetric polymerase chain reaction and gold silicon particles

A new paper-based lateral flow nucleic acid (LFNA) test platform was established in this study using asymmetric polymerase chain ceaction (A-PCR) for signal amplification. This new method allowed a visual detection of Epstein-Barr virus (EBV) nucleic acids with high specificity and low cost. In addition, as part of our strategy we employed a sandwich system of capture probe (CP)/gold nanoparticles (AuNPS) and silicon dioxide (SiO2) (AuNPS@SiO2) nanospheres/target DNA/avidin complexes as the sensing platform. Biotin-labeled target DNA was obtained by A-PCR and later introduced in the LF device. The CP/target DNA/AuNPS@SiO2 complexes were captured on the test zone by the specific reaction between biotin and avidin, and the remaining CP/AuNPS@SiO2 particles were captured on the quality control zone by the hybridization between CP and a quality control probe. Au@SiO2 accumulation in the test and quality control zones of the device enabled a visual detection of the specific target sequences. The method detection limit was 50 nM of the target DNA, which was lower than that of the LFNA biosensor (LFNAB) without PCR amplification and Au@SiO2 particles. In conclusion, the novel paper-based platform described here is a low cost, efficient and fast visual detection method that offers high sensitivity and other benefits compared to alternative methods in use.

Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor.

Genome-editing techniques incorporating artificial nucleases develop rapidly and enable efficient and precise modification of genomic DNA of numerous organisms. The present researchaimed to establish a rapid, sensitive and visual method for genotyping of germline genome-editedmutants with small genomic fragment deletion. The genome-edited pigs with 2-bp deletion and 11-bp deletion of Myostatin (MSTN) gene generated by TALENs system were used as test materials to check the proposed allele-specific PCR (AS-PCR) and lateral flow nucleic acid biosensor (LFNAB) cascade method. AS-PCR can produce products with different tags to distinguish genome-edited alleles and wild-type alleles. A LFNAB was applied to do visual detection of AS-PCR products without using additional instruments. Furthermore, we demonstrated that AS-PCR and LFNAB cascade could accurately and visually distinguish genome-edited pigs with small genomic fragment deletion of Myostatin (MSTN) gene and wild-type pigs with limitof detection (LOD) of 0.1 ng. The proposed AS-PCR and LFNAB cascade can do rapid and visual genotyping of genome-editedmutants with small genomic fragment deletion, serving as a platform for genome-edited animal genotyping.

Gold nanorods-based lateral flow biosensors for sensitive detection of nucleic acids.

Agold nanorod (AuNR)-based lateral flow nucleic acid biosensor (LFNAB) is reportedfor visual detection of DNA with a short test time and high sensitivity. AuNRs with an approximate length of 60nm were utilized as a colored tag to label the detection DNA probe (Det-DNA). The capture DNA probe (Cap-DNA) was immobilized on the test region of LFNAB. Sandwich-type complex was formed among the AuNR-Det-DNA, target DNA (Tar-DNA), and Cap-DNA on the LFNAB by Watson-Crick base pairing. In the presence of Tar-DNA, AuNRs were thus seized on the test region of LFNAB, and the accumulation of AuNRs subsequently produced a characteristic colored band. The optimized LFNAB was able to detect 10 pM Tar-DNA without instrumentation. Quantitative analysis could be established by measuring the intensity of test band using a portable strip reader, and the detection limit of 2 pM target DNA was achieved on the LFNAB without signal amplification. The detection limit of the AuNR-based LFNAB is 250-fold lower than that of gold nanoparticle (AuNP)-based LFNABs. This work unveiled a sensitive, rapid, and economical strategy for the detection of nucleic acids, and simultaneously opening new promising routesfor disease diagnosis and clinical applications. Gold nanorods are used as colored tags for lateral flow nucleic acid biosensor.

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.

16S rRNA-functionalized multi-HCR concatemers in a signal amplification nanostructure for visual detection of Salmonella.

To prevent foodborne diseases and minimize their impacts, it is extremely important to develop a cost-effective and efficient bacterial detection assay for diagnostics, particularly in resource-poor settings. In this study, 16S rRNA from foodborne Salmonella was coupled with multiple HCR (hybridization chain reaction) concatemers and functionalized in a signal structure for lateral flow nucleic acid biosensor (LFNAB) detection. The 16S rRNA was incubated with two specific capture probes and multiple helper probes carrying the same initiator, to unwind its secondary structure and form an "initiators-on-a-string" complex. Through use of the initiators, each target 16S rRNA yielded multiple HCR concatemers tethered to numerous biotins, and numerous streptavidin-labeled gold nanoparticles were introduced on the LFNAB. The limit of detection was 53.65 CFU/mL for Salmonella. Notably, this method has high specificity and applicability for the detection of Salmonella in food and water samples.

Point-of-care detection of 16S rRNA of Staphylococcus aureus based on multiple biotin-labeled DNA probes

A visual method that combines multiple biotin-labeled DNA probes and lateral-flow nucleic acid biosensor was developed to detect Staphylococcus aureus. The 16S rRNA from Staphyloccocus aureus (S. aureus), coupled with multiple biotin-labeled DNA probes, was functionalized in a signal structure for lateral-flow point-of-care detection. The secondary structure of the 16S rRNA was unwound by two specific capture probes modified by Fam and multiple bridge probes, which extended additional sequences for use as initiators. By utilizing the initiators, each target 16S rRNA with multiple DNA probes could tether a number of biotin molecules, so that a large number of streptavidin-labeled gold nanoparticles could be introduced in the lateral flow assay. The images of the lateral flow detection results obtained using a smartphone were transmitted to a computer via Wi-Fi or Bluetooth connection for quantitative processing by ImageJ. The limit of detection was 103 cfu/mL without sample enrichment, and decreased to 0.12 cfu/mL following a 3-h enrichment of samples in growth medium. Notably, this method presented high specificity and applicability for the detection of S. aureus in food samples. In short, the developed visual non-specific operation method is very suitable for point-of-care diagnosis of pathogens in resource-limited countries.

Lateral flow test for visual detection of multiple MicroRNAs

The authors describe a rapid and low-cost approach for multiplex microRNA(miRNA) assay on lateral flow nucleic acid biosensor (LFNAB). The principle of assay is based on sandwich-type nucleic acid hybridization reactions to produce gold nanoparticle (GNP)-attached complexes (ssDNA-microRNA-ssDNA/GNPs), which are captured and visualized on the test zone of LFNAB. By designing three different test zones on LFNAB, simultaneous detection of microRNA-21, microRNA-155 and microRNA-210 was achieved with an adding-measuring model by using GNP as visual tag. The method was challenged by testing the microRNAs in spiked serum samples with satisfied results. In our perception, the test is a particularly valuable tool for clinical application and biomedical diagnosis, particularly in limited resource settings.

Lateral flow nucleic acid biosensor for sensitive detection of microRNAs based on the dual amplification strategy of duplex-specific nuclease and hybridization chain reaction.

MicroRNAs (miRNAs) constitute novel biomarkers for various diseases. Accurate and quantitative analysis of miRNA expression is critical for biomedical research and clinical theranostics. In this study, a method was developed for sensitive and specific detection of miRNAs via dual signal amplification based on duplex specific nuclease (DSN) and hybridization chain reaction (HCR). A reporter probe (RP), comprising recognition sequence (3’ end modified with biotin) for a target miRNA of miR-21 and capture sequence (5’ end modified with Fam) for HCR product, was designed and synthesized. HCR was initiated by partial sequence of initiator probe (IP), the other part of which can hybridize with capture sequence of RP, and was assembled by hairpin probes modified with biotin (H1-bio and H2-bio). A miR-21 triggered cyclical DSN cleavage of RP, which was immobilized to a streptavidin (SA) coated magnetic bead (MB). The released Fam labeled capture sequence then hybridized with the HCR product to generate a detectable dsDNA. This polymer was then dropped on lateral flow strip and positive result was observed. The proposed method allowed quantitative sequence-specific detection of miR-21 (with a detection limit of 2.1 fM, S/N = 3) in a dynamic range from 100 fM to 100 pM, with an excellent ability to discriminate differences in miRNAs. The method showed acceptable testing recoveries for the determination of miRNAs in serum.

Visual detection of nucleic acids based on lateral flow biosensor and hybridization chain reaction amplification

In this study, a new lateral flow nucleic acid biosensor (LFNAB) using hybridization chain reaction (HCR) for signal amplification was developed for visual detection of nucleic acids with high sensitivity and low cost. A “sandwich-type” detection strategy was employed in our design. The sandwich system of capture probe (CP)/target DNA/reporter probe (RP)-HCR complexes was fabricated as the sensing platform. As the initiator strand, reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin, and determined whether long nicked DNA polymers were formed. The biotin-labeled double-strand DNA polymers then introduced numerous Streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the lateral flow device. The CP/target DNA/RP-HCR complexes were captured on the test zone by the specific reaction between anti-Fam monoclonal antibody (anti-Fam mAb) on the test zone and Fam of the complexes. The accumulation of AuNPs on the test zone of the biosensor enabled the visual detection of specific sequences. The detection limit of specific DNA was as low as 1.76pM, which was about 2 orders lower than that of the LFNAB without HCR amplification. And the detection limit of Salmonella was 3×103cfumL−1. In conclusion, this visual detection system, HCR-LFNAB, is suitable for non-specialist personnel and point-of-care (POC) diagnosis in low-resource settings.

A three-line lateral flow biosensor for logic detection of microRNA based on Y-shaped junction DNA and target recycling amplification

A rapid, sensitive, and accurate detection strategy for microRNA 16 (miR-16) was developed, which combined the convenience of lateral flow biosensors (LFBs), the design flexibility of Y-shaped junction DNA probe, and the enhancement ability of endonuclease-assisted target recycling amplification. The systemis composed of a molecular beacon (MB) probe, an assistant probe, and endonuclease Nt.BbvCI, which plays the role of signal translation and amplification. In the presence of the target microRNAs (miRNAs), three chains of nucleic acid could hybridize with each other to form a Y-shaped junction structure, which could be recognized by the endonuclease Nt.BbvCI. The MB probe was efficiently cleaved by endonuclease and produced two new DNA fragments, while the regenerated assistant probe and target were hybridized to another MB probe and entered into the next cycle of the amplification. In this way, the detection of the readily biodegradable miRNA was turned into the detection of two DNA fragments in the LFB. Meanwhile, the detection of two different DNAs would improve the accuracy and effectively avoid false results. The amplified products containing DNA fragments were then applied to the lateral flow nucleic acid biosensor (LFNAB) with two test zones, on which specific DNA probes were designed. The formed DNA-DNA/gold nanoparticle (GNP) conjugates were captured and accumulated to produce two red bands in two test zones. The logic judgment of the two test zones provided more accurate and convincing results. Under optimal conditions, the visual detection limit of miR-16 in aqueous solutions was 0.1pM, which is 100-1000 times lower than that of visual or colorimetric methods in the literature. It could be used for on-field and point-of-care testing and meet the urgent demand of sensitive and selective miRNA detection in remote rural areas without costly equipment. The system displayed good universality, compatibility, high specificity, and stability of miRNA detection, which revealed significant potentiality in biomedical diagnosis. Graphical abstract A simple and rapid approach for microRNA-16 (miR-16) detection was developed based on Y-shaped junction DNA probe and the endonuclease-assisted target recycling amplification using lateral flow biosensors.

Gold Nanoparticle Coated Silica Nanorods for Sensitive Visual Detection of microRNA on a Lateral Flow Strip Biosensor.

We present a rapid and highly sensitive approach for visual detection of microRNA (miRNA) using a gold nanoparticles coated silica nanorod label and lateral flow strip biosensor. Gold nanoparticles were decorated on the silica nanorod surface by a seeding and growth procedure. A single strand DNA probe was immobilized on the gold nanoparticles-silica nanorod surface by a self-assembling process, and the formed DNA-gold nanoparticles-silica nanorod conjugate was used to construct the lateral flow nucleic acid biosensor for detecting miRNA. The captured gold nanoparticles-silica nanorods by sandwich-type hybridization reactions (DNA-RNA-DNA) on the test zone of the lateral flow nucleic acid biosensor produced the characteristic color bands, enabling visual detection of miRNA. After systematic optimization, the new lateral flow nucleic acid biosensor was capable of detecting 10 pM of the miRNA target without instrumentation, which is six times lower than that obtained with the gold nanoparticle-based lateral flow nucleic acid biosensor. The gold nanoparticles coated silica nanorod thus provides a new and sensitive nanolabel for visual detection of biological molecules on the lateral flow biosensor.

Proficient Detection of Multi-Drug-Resistant Mycobacterium tuberculosis by Padlock Probes and Lateral Flow Nucleic Acid Biosensors.

Tuberculosis is a major communicable disease. Its causative agent, Mycobacterium tuberculosis, becomes resistant to antibiotics by acquisition of point mutations in the chromosome. Multi-drug-resistant tuberculosis (MDR-TB) is an increasing public health threat, and prompt detection of such strains is of critical importance. As rolling circle amplification of padlock probes can be used to robustly distinguish single-nucleotide variants, we combined this technique with a sensitive lateral flow nucleic acid biosensor to develop a rapid molecular diagnostic test for MDR-TB. A proof-of-concept test was established for detection of the most common mutations [rpoB 531 (TCG/TTG) and katG 315 (AGC/ACC)] causing MDR-TB and verification of loss of the respective wild type. The molecular diagnostic test produces visual signals corresponding to the respective genotypes on lateral flow strips in approximately 75 min. By detecting only two mutations, the test can detect about 60% of all MDR-TB cases. The padlock probe-lateral flow (PLP-LF) test is the first of its kind and can ideally be performed at resource-limited clinical laboratories. Rapid information about the drug-susceptibility pattern can assist clinicians to choose suitable treatment regimens and take appropriate infection control actions rather than prescribing empirical treatment, thereby helping to control the spread of MDR-TB in the community.

Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor

This study describes a simple and sensitive approach for visual and point-of-care detection of P. aeruginosa and its toxin genes based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow nucleic acid biosensor (LFNAB). Differentiation of the internal standard gene ecfX and toxin genes (ExoS and ExoU) in P. aeruginosa was determined using FITC-, hex-and digoxin-modified primers in the mLAMP process. In the presence of biotin-and FITC- (hex-, digoxin-) modified primers and Bst DNA polymerase large fragments, the mLAMP produced numerous biotin- and FITC- (hex-, digoxin-) attached duplex DNA products. The products were detected by LFNAB through dual immunoreactions (anti-biotin antibodies on the gold nanoparticle (Au-NP) and biotin on the duplex, anti-FITC (hex, digoxin) antibodies on the LFNAB test line and FITC (hex, digoxin) on the duplex). The accumulation of Au-NPs produced a characteristic red band, enabling visual detection of P. aeruginosa and its toxin genes without instrumentation. After systematic optimization of LFNAB preparation and detecting conditions, the current approach was capable of detecting concentrations as low as 20 CFU/mL P. aeruginosa or its toxin genes within 50min without complicated instrument, which is more sensitive than PCR. Therefore, this approach provides a simple, pollution free, sensitive, and low-cost point-of-care test for the detection of P. aeruginosa and its toxin genes.

Accurate and easy-to-use assessment of contiguous DNA methylation sites based on proportion competitive quantitative-PCR and lateral flow nucleic acid biosensor

Many types of diagnostic technologies have been reported for DNA methylation, but they require a standard curve for quantification or only show moderate accuracy. Moreover, most technologies have difficulty providing information on the level of methylation at specific contiguous multi-sites, not to mention easy-to-use detection to eliminate labor-intensive procedures. We have addressed these limitations and report here a cascade strategy that combines proportion competitive quantitative PCR (PCQ-PCR) and lateral flow nucleic acid biosensor (LFNAB), resulting in accurate and easy-to-use assessment. The P16 gene with specific multi-methylated sites, a well-studied tumor suppressor gene, was used as the target DNA sequence model. First, PCQ-PCR provided amplification products with an accurate proportion of multi-methylated sites following the principle of proportionality, and double-labeled duplex DNA was synthesized. Then, a LFNAB strategy was further employed for amplified signal detection via immune affinity recognition, and the exact level of site-specific methylation could be determined by the relative intensity of the test line and internal reference line. This combination resulted in all recoveries being greater than 94%, which are pretty satisfactory recoveries in DNA methylation assessment. Moreover, the developed cascades show significantly high usability as a simple, sensitive, and low-cost tool. Therefore, as a universal platform for sensing systems for the detection of contiguous multi-sites of DNA methylation without external standards and expensive instrumentation, this PCQ-PCR-LFNAB cascade method shows great promise for the point-of-care diagnosis of cancer risk and therapeutics.

Detection of isoniazid-resistant Mycobacterium tuberculosis by padlock probes combined with lateral flow nucleic acid biosensor

Detection of isoniazid-resistant Mycobacterium tuberculosis by padlock probes combined with lateral flow nucleic acid biosensor

Visual detection of microRNA with lateral flow nucleic acid biosensor

We report a DNA-gold nanoparticle (DNA-GNP) based lateral flow nucleic acid biosensor for visual detection of microRNA (miRNA)-215 in aqueous solutions and biological samples with low-cost and short analysis time. Sandwich-type hybridization reactions among GNP-labeled DNA probe, miRNA-215 and biotin-modified DNA probes were performed on the lateral flow device. The accumulation of GNPs on the test zone of the biosensor enables the visual detection of miRNA-215. After systematic optimization, the biosensor was able to detect a minimum concentration of 60pM miRNA-215. The biosensor was applied to detect miRNA-215 from A549 cell lysate directly without complex sample treatment, and the detection limit of 0.148 million cells was obtained. This study provides a simple, rapid, specific and low-cost approach for miRNA detection in aqueous solutions and biological samples, showing great promise for clinical application and biomedical diagnosis in some malignant diseases.

Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia

A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.

Dry-reagent nucleic acid biosensor based on blue dye doped latex beads and lateral flow strip

In the manuscript, a quantitative lateral flow nucleic acid biosensor (Lateral flow nucleic acid biosensor, LFNB) based on blue dye doped latex beads was proposed and its feasibility for detecting deoxyribonucleic acid (DNA) in plasma was investigated. A 60-mer DNA sequence (T1) was selected as model to demonstrate the protocol. Blue dyes doped latex bead bearing DNA probe would be captured on the corresponding test line in the presence of target DNA, to form an evident blue band. Although qualitative tests are realized by observing the color change of the test zone, quantitative data are obtained by recording the optical responses of the test zone with a portable “Strip Reader” instrument conveniently. The strip has been applied for the detection of synthesized DNA sample in human plasma sample with a detection limit of 3.75fmol. Interference was not evident even the target DNA was spiked with 50μL plasma which indicated the well shielding of the latex bead reporters and quantified chromatographic separations of unwanted materials of the strip comparing with traditional gold nanoparticle based LFNB platforms.