Lateral Flow Biosensor Research Articles

Rapid, on-site and yes-or-no detection of Salmonella typhimurium in foods using Argonaute-enabled assay

Early detection and control of foodborne diseases are crucial for ensuring food safety and consumer health. Therefore, there is a need for faster and more sensitive detection methods. We developed a rapid, portable and visible lateral flow biosensor (LFB) for foodborne pathogenic bacteria detection powered by a Pyrococcus furiosus Argonaute nuclease (PfAgo), named as PfAgo-LFB. LAMP (Loop-mediated isothermal amplification) was used to amplify the target nucleic acid. By using PfAgo as the “lighting up” technology, we can overcome the limitations of false-positive results and lack of effective endpoint monitoring in LAMP, greatly enhancing its applicability. It turned out that PfAgo-LFB was able to provide yes-or-no (positive or negative) results with a limit of detection (LOD) as low as 1 CFU/mL for S. typhimurium and the detection range was between 100 to 108 CFU/mL. It was able to secure a sample-to-answer test for contaminated food samples in less than 45 min and demonstrated satisfactory selectivity. In summary, we designed a PfAgo-LFB that rendered a novel, rapid, accurate, easy-to-deploy, on-site and yes or no detection of S. typhimurium, which could be generalized for building a “one-for-all” biosensing platform.

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Development point-of-care based lateral flow biosensor for the rapid detection of exosomes of subarachnoid hemorrhage patients

Exosomes are extracellular vesicles with diameters ranging from 30–200 nm, and the biomolecules contained in exosomes have been used as biomarkers for the diagnosis and prognosis of certain diseases.

MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples.

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

An Aptamer-Based Lateral Flow Biosensor for Low-Cost, Rapid and Instrument-Free Detection of Ochratoxin A in Food Samples.

In this work, a simple and cost-efficient aptasensor strip is developed for the rapid detection of OTA in food samples. The biosensor is based on the lateral flow assay concept using an OTA-specific aptamer for biorecognition of the target analyte. The strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane (NC) and an absorbent pad. The conjugate pad is loaded with the OTA-specific aptamer conjugated with gold nanoparticles (AuNPs). The test line of the NC membrane is loaded with a specific OTA-aptamer probe and the control line is loaded with a control probe. The assay is based on a competitive format, where the OTA present in the sample combines with the OTA aptamer-AuNP conjugate and prevents the interaction between the specific probe immobilized on the test line and the OTA aptamer-AuNP conjugates; therefore, the color intensity of the test line decreases as the concentration of OTA in the sample increases. Qualitative detection of OTA is performed visually, while quantification is performed by reflectance colorimetry using a commercial scanner and image analysis. All the parameters of the assay are investigated in detail and the analytical features are established. The visual limit of detection (LOD) of the strip is 0.05 ng mL-1, while the LOD for semi-quantitative detection using reflectance colorimetry is 0.02 ng mL-1. The lateral flow strip aptasensor is applied to the detection of OTA in wine, beer, apple juice and milk samples with recoveries in the range from 91 to 114%. The assay exhibits a satisfactory selectivity for OTA with respect to other mycotoxins and lasts 20 min. Therefore, the lateral flow strip aptasensor could be useful for the rapid, low-cost and fit-for-purpose on-site detection of OTA in food samples.

Rapid, sensitive and highly specific diagnosis of Moraxella catarrhalis by recombinase polymerase amplification-based biosensor and fluorescence detection

Moraxella catarrhalis (M. catarrhalis) was an underestimated respiratory infection pathogen that has been largely overlooked. The limited availability of rapid and sensitive detection methodologies has hindered M. catarrhalis diagnostic in clinical settings and contributed to its underestimation. To address this issue, we devised two recombinase polymerase amplification (RPA)-based assays for rapid, sensitive and reliable detection of M. catarrhalis, termed M. catarrhalis-RPA-Flu and M. catarrhalis-RPA-LFB, which utilized fluorescence and nanoparticle-based lateral flow biosensor (LFB) for reporting the detection results, respectively. In both assays, the specific copB gene of M. catarrhalis was amplified at 37°C for only a period of 20 minutes. In M. catarrhalis-RPA-Flu system, the detection results were analyzed by either using a real-time fluorescent detector or by direct observation using the naked eye under blue light, while, in M. catarrhalis-RPA-LFB system, biosensors were used for interpreting the results without any specialized instruments. Both methods were able to finalize the entire detection process within a duration of 40 minutes, detect down to 35 fg genomic DNA per test, and correctly differentiate M. catarrhalis from non-M. catarrhalis strains. The feasibility of both techniques was validated by analyzing 96 BALF (Broncho alveolar lavage fluid) samples in clinical settings. Collectively, the newly developed two RPA-based assays exhibit great potential for rapid and accurate identification of M. catarrhalis in standard microbiology laboratories as well as diagnosis of M. catarrhalis infection in clinical settings.

Loop-mediated isothermal amplification coupled with nanoparticle-based lateral flow biosensor for monkeypox virus detection

Monkeypox virus (MPXV) infection is currently an evolving public health concern, highlighting an urgent need for early and rapid detection of MPXV. Here, we present a diagnostic test called MPXV-LAMP-LFB, which combines loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor (LFB) for the simple, sensitive and specific detection of MPXV and differentiation of its two clades. The MPXV-LAMP-LFB can be conducted at a heating block and the detection results can be visually indicated with the biosensor without any specialized apparatus. Two sets of LAMP primers targeting the D14L and ATI genes were designed for the Central and West African MPXV isolates, respectively. The optimal amplification condition was 64 °C for 40 min. Thus, the MPXV-LAMP-LFB test can be completed within 1 h, incorporating rapid DNA extraction (∼15 min), LAMP reaction (∼40 min) and result indicating (∼5 min). The MPXV-LAMP-LFB assay could detect down to 5 copies of plasmid template and 12.5 copies of pseudotyped virus in simulated blood samples. Furthermore, the MPXV-LAMP-LFB assay correctly identified all the positive controls and successfully avoided cross-reactivity with the non-MPXV pathogens or clinical samples, demonstrating its high specificity. Overall, the MPXV-LAMP-LFB test developed in this study showed great promise as a rapid, sensitive and accurate molecular tool for diagnosing MPXV infection.

Rapid and sensitive detection of Pseudomonas aeruginosa by isothermal amplification combined with Cas12a-mediated detection

CRISPR based technologies have been used for fast and sensitive detection of pathogens. To test the possibility of CRISPR based detection strategy in Pseudomonas aeruginosa infections, a combined method of recombinase polymerase amplification followed by Cas12a-mediated detection via fluorescence reader or lateral flow biosensor (named Cas12a-RCFL) has been established in this study. The Cas12a-RCFL can detect as low as 50 CFU/mL Pseudomonas aeruginosa. The whole detection process can be finished within one hour with satisfied detection specificity. Cas12a-RCFL also shows good sensitivity of detecting Pseudomonas aeruginosa inStaphylococcus aureus and Acinetobacter baumannii contaminated samples. For the detection of 22 clinical samples, Cas12a-RCFL matches with PCR sequencing result exactly without DNA purification. This Cas12a-RCFL is rapid and sensitive with low cost, which shows good quality to be adopted as a point-of-care testing method.

Zn2+/GNPs nanocomposite for highly selective colorimetric detection of creatinine in urine samples of CKD patients

This research reports the fabrication of Zn2+/gold nanoparticles (GNPs) nanocomposite for colorimetric detection of creatinine (CR) in diverse media (e.g., water, artificial urine, and urine samples) between healthy subjects and chronic kidney disease (CKD) patients. The color of GNPs in suspension changed from characteristic wine-red (λabsorption = 545 ± 5 nm) to colorless or black upon the formation of nanocomposite with Zn2+ ions. However, upon addition of CR to Zn2+/GNPs nanocomposite suspension, the characteristic wine-red color of GNPs was restored. Our experimental analysis evidently validated the hypothesis on Zn2+ induced agglomeration of GNPs and their subsequent anti-agglomeration upon the addition of CR to the Zn2+/GNPs. Overall, our approach offered highly sensitive recognition of CR with a limit of detection of 2 µg·mL−1 (R2 = 0.95) along with excellent stability (>three months), selectivity (in the presence of interfering biochemicals (e.g., urea,ascorbic acid, and glucose, glutathione, Na+, K+, Ca2+, Mg2+, PO43−, and SO4 2−)), and reproducibility (relative standard deviation ∼ 9 %). Finally, the great potential of our method for CR recognition was also confirmed by good agreement (R2 = 0.95) with the gold standard ‘Jaffe’ method along with the Bland-Altman analysis for urine samples between health subjects (n = 15) and CKD patients (n = 11). In near future, a quantitative lateral flow biosensor is expected to be developed for non-invasive detection of CR through the integration of the proposed approach with the machine learning tools.

Decorated DNA-Based Scaffolds as Lateral Flow Biosensors.

Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have rationally re-engineered tile-based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof-of-principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti-Dig, Anti-DNP and Anti-MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA-based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity.

Decorated DNA‐Based Scaffolds as Lateral Flow Biosensors

AbstractHere we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA‐based structures decorated with reporter tags and recognition elements. We have rationally re‐engineered tile‐based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof‐of‐principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti‐Dig, Anti‐DNP and Anti‐MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA‐based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity.

A modified multiple cross displacement amplification linked with a gold nanoparticle biosensor for the detection of Epstein-Barr virus in clinical applications.

Epstein-Barr virus (EBV), a double-stranded DNA virus belonging to the family Herpesviridae, infects more than 95% of healthy adults by attacking the host immune system. Here, a novel detection protocol, utilizing the modified multiple cross displacement amplification (MCDA) technique combined with a gold nanoparticles-based lateral flow biosensors (AuNPs-LFB), was devised and developed to detect EBV infection (termed EBV-MCDA-LFB assay). Ten MCDA primers targeting the EBNA-LP gene were designed, including CP1* primers modified with 6-carboxyfluorescein (FAM) and D1* primers modified with biotin. Then, nucleic acid templates extracted from various pathogens and whole blood samples were used to optimize and evaluate the EBV-MCDA-LFB assay. As a result, the lowest concentration of EBNA-plasmids, which can be detected by MCDA-LFB assay with an optimal reaction condition of 67°C for 30 min, was 10 copies/reaction. Here, the MCDA-LFB assay can detect all EBV pathogens used in the study, and no cross-reactions with non-EBV organisms were observed. Meanwhile, the entire detection workflow of the EBV-MCDA-LFB assay for whole blood samples, including DNA template preparation (25 min), EBV-MCDA amplification (30 min), and AuNPs-LFB-mediated validation (2-5 min), can be completed within 1 h. Taken together, the EBV-MCDA-LFB assay established in the current study is a rapid, simplified, sensitive, specific, and easy-to-obtain technique that can be used as a screening or diagnostic tool for EBV infection in clinical applications, especially in resource-poor regions.

Biosensor-Based Multiple Cross Displacement Amplification for the Rapid Detection of Mycobacterium leprae.

Leprosy is an ancient disease caused by Mycobacterium leprae (ML) that remains a public health problem in poverty-stricken areas worldwide. Although many ML detection techniques have been used, a rapid and sensitive tool is essential for the early detection and treatment of leprosy. Herein, we developed a rapid ML detection technique by combining multiple cross displacement amplification (MCDA) with a nanoparticle-based lateral flow biosensor (LFB), termed ML-MCDA-LFB. MCDA induced a rapid isothermal reaction using specific primers targeting the RLEP gene, and the LFB enabled instant visual amplicon detection. The pure genomic DNA of ML and nucleic acids from various pathogens were employed to evaluate and optimize the ML-MCDA-LFB assay. The optimal conditions for ML-MCDA-LFB were 68 °C and 35 min, respectively. The limit of detection for pure ML genomic DNA was 150 fg per vessel, and the specificity of detection was 100% for the experimental strains. Additionally, the entire detection process could be performed within 40 min, including the isothermal amplification (35 min) and result confirmation (1-2 min). Hence, the ML-MCDA-LFB assay was shown to be a rapid, sensitive, and visual method for detecting ML and could be used as a potential tool for early clinical diagnosis and field screening of leprosy.

Using dCas9 as an intermediate bridge of loop-mediated isothermal amplification-based lateral flow colorimetric biosensor for point-of-care Salmonella detection

Foodborne pathogens pose a serious threat to human health worldwide. Combining isothermal amplification techniques (e.g., loop-mediated isothermal amplification, LAMP) with lateral flow biosensor (LFB) is ideal for point-of-care testing of foodborne pathogens, but challenges remain in distinguishing target amplicons and by-products. Here, we developed a portable lateral flow colorimetric biosensor, termed CALL (dCas9-assisted LAMP-based LFB), using dCas9 as an intermediate bridge to address these challenges. As a proof-of-concept, we chose Salmonella as the model. Strategically, LAMP amplicons of the target gene contain many contiguous repeats that can be recognized and unlocked by dCas9/single-guide RNA, which then serve as scaffolds to assemble many gold nanoparticles (GNPs) into chainlike structures on the test line. This strategy eliminates false positives caused by by-products and enables all repeats of LAMP amplicon to participate in capturing GNPs, greatly improving specificity and sensitivity. The biosensor integrates rapid extraction, LAMP amplification, dCas9-based assembly and LFB analysis without requiring complex equipment, enabling on-site screening of Salmonella as low as 41 CFU/mL in 40 min. The practicability of the biosensor was further demonstrated by testing real foods. The success of CALL provides a promising direction for developing foodborne pathogen detection tools.

Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex.

Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1h. The limit of detection (LoD) was 4 copies μL-1, and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.

Optimization of a rapid and sensitive nucleic acid lateral flow biosensor for hepatitis B virus detection.

The utilization of direct amplification of nucleic acid from lysate has attracted interest in the advancement of straightforward and economical point-of-care assays. Consequently, this study primarily focuses on the development of a rapid, precise, and cost-effective lateral flow biosensor for the convenient detection of HBV nucleic acid at the point-of-care. Furthermore, the study evaluates the effectiveness of the direct amplification method in comparison to purified nucleic acid samples within the context of LAMP-LF biosensing approaches. The experiments conducted in this study utilized clinical serum samples that were confirmed as HBV-positive through real-time PCR assays. Sample preparation involved employing spin column nucleic acid purification and serum heat treatment. To amplify a 250bp fragment of the HBV polymerase gene, three pairs of specific LAMP primers were utilized, which were biotin-labeled and FITC-labeled for detection purposes. Various incubation temperatures (ranging from 64 to 68°C) and durations (30min, 45min, and 1h) were investigated to determine the optimal conditions for the LAMP assay. The results were subsequently assessed through fluorometric analysis, white turbidity measurements, and lateral flow assay. Milenia HybriDetect1 strips, designed for immediate use, were employed to visualize the LAMP amplicons. Furthermore, the performance of the lateral flow biosensor was evaluated using 10-fold serial dilutions of a secondary standard containing a viral load of 108 IU/ml. The optimization of the LAMP reaction was achieved at a temperature of 67°C, resulting in significant turbidity after a 30-minute incubation period. When the spin column purification method was employed, varying test bands were observed for templates ranging from 108 IU/ml to 101 IU/ml viral load. However, when serum samples underwent heat treatment and the resulting supernatant was directly used for LAMP, the lateral flow assay was capable of detecting a minimum viral load of 103 IU/ml. In resource-limited settings, the LAMP-LF assay presents a promising solution for HBV testing. However, it is important to note that direct amplification without DNA purification may diminish the performance of the approach.

Gold nanoparticle assisted colorimetric biosensors for rapid polyethylene terephthalate (PET) sensing for sustainable environment to monitor microplastics

The extremely widespread and ubiquitous nature of plastics, estimated to boost its global production by 26 billion tons till 2050. The large chunks of plastic waste that decomposed down to micro- or nano plastics (MNPs) leads to various ill effects on biological entities. The conventional PET detection methods lack rapid detection of microplastics due to variances in microplastic features, long-drawn-out sample pre-processing procedures and complex instrumentation. Therefore, an instantaneous colorimetric evaluation of microplastic will ensures the simplicity of conducting assays on field. Several nanoparticle-based biosensors that detects proteins, nucleic acids, metabolites operate on either cluster or disperse state of nanoparticle. However, gold nanoparticle (AuNPs) emerges an ideal scaffold for sensory element in lateral flow biosensors due to their simple surface functionalization, unique optoelectronic properties and varied colour spectrum depending on morphologies and aggregation state. In this paper an effort has been made in the form of a hypothesis using in silico tools as a basis to detect polyethylene terephthalate (PET) – most abundant type of microplastic using gold nanoparticle based lateral flow biosensor. We retrieved sequences of PET-binding synthetic peptides and modelled their 3-D structure using I-Tasser server. The best protein model for each peptide sequences are docked with PET monomers – BHET, MHET and other PET polymeric ligands, to evaluate their binding affinities. The synthetic peptide SP 1 (WPAWKTHPILRM) docked with BHET and (MHET)4 exhibits 1.5-fold increases in binding affinity as compared to reference PET anchor peptide Dermaseptin SI (DSI). The GROMACS molecular dynamics simulation studies of synthetic peptide SP 1 - BHET & - (MHET)4 complexes for 50 ns further confirmed the stable binding. RMSF, RMSD, hydrogen bonds, Rg and SASA analysis provides useful structural insights of the SP 1 complexes as compared to reference DSI. Furthermore, SP 1 functionalized AuNP-based colorimetric device was described in detail for detection of PET.

State-of-the-art of portable (bio)sensors based on smartphone, lateral flow and microfluidics systems in protozoan parasites monitoring: A review

Up to now, the importance of different analytical methods has increased more and more in terms of the detection of various targets, protozoan parasites included. Despite the advancement of several sensing approaches for detection of them, the development of portable biosensors which can provide point-of-care detection can be considered the next generation of sensing platforms. Among these, smartphone-based bio(sensors), microfluidics systems, and lateral flow biosensors have attracted great attention. To elaborate, all of them have a specific ability to turn into an efficient analytical approach. The accessibility of smartphones can mature a simple communication gadget into an opportunity for easy use sensing device, and alongside that, the small size of the microfluidics system can provide a miniature analytical method that consumes a low number of materials and, in turn, it can bring about the affordable platform. In favor of lateral flow biosensors, the undeniable advantages of this method including low-cost, user-friendly, and in-field diagnosis with good robustness can introduce them as an efficient sensing platform. Hence, the review of the state-of-the-art of portable (bio)sensors based on smartphone, lateral flow, and microfluidics systems can broaden persons’ horizons about novel sensing approaches which can be used for the determination of protozoa parasites. Furthermore, the role of nanomaterials, their benefits, and their drawbacks will be discussed in the following.

Ultrasensitive lateral flow biosensor based on PtAu@CNTs nanocomposite catalytic chromogenic signal amplification strategy for the detection of nucleic acid

A rapid and ultrasensitive lateral flow biosensor was developed, which based on gold and platinum nanoparticles-decorated carbon nanotubes (PtAu@CNTs) nanocomposite catalytic chromogenic signal amplification strategy for the detection of nucleic acid. Independent platinum and gold nanoparticles modified functional carbon nanotubes (PtAu@CNTs) were prepared by in-situ reduction. Sandwich-type hybridization reaction occurred between PtAu@CNTs-labeled DNA probe, target DNA and Biotin-modified DNA probes, which was captured on test zone of the strip. Accumulation of PtAu@CNTs nano-labels formed a characteristic colored band. After systematic optimization and catalytic chromogen, the naked eye detection limit of PtAu@CNTs-LFA was about 2 pM, and the theoretical detection limit of target DNA is calculated to be 0.43 pM according to the standard curve. The results indicates a rapid, sensitive and specific methods for DNA detection in biological samples, showing great promise for biomedical diagnosis in some malignant diseases in clinical application.

CRISPR/Cas9-based point-of-care lateral flow biosensor with improved performance for rapid and robust detection of Mycoplasma pneumonia

Screening of acute respiratory infections causes serious challenges in urgent point-of-care scenarios where conventional methods are impractical and alternative techniques suffer from low accuracy, poor robustness, and reliance on sophisticated instruments. As an improvement to this paradigm, we report a point-of-care lateral flow biosensor (LFB) based on the recognition property of clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (Cas9) and apply it to the detection of Mycoplasma pneumoniae (M. pneumoniae). The designed biosensor employs CRISPR/Cas9 for secondary recognition after preamplification of target gene using specific primer set, avoiding false positives caused by nontarget factors. The high amplification efficiency and low applicable temperatures of recombinase polymerase amplification brings the detection limit of the biosensor to 3 copies even at a preamplification temperature of 25 °C. Its practical application is further demonstrated with 100% accuracy by testing with 43 M. pneumoniae-infected specimens and 80 uninfected specimens. Additionally, the entire detection, including pretreatment, preamplification, CRISPR/Cas9 recognition, and visual analysis, can be completed in 30 min. Featured with the combination of CRISPR/Cas9 and LFB, the biosensor we developed herein ensures excellent convenience, accuracy, and robustness, which endows promising point-of-care screening potential for infectious pathogens.