An immunochromatographic test strip for on-site detection of Ralstonia solanacearum in tobacco leaves and soil.

Tobacco bacterial wilt is one of the most devastating soil-borne diseases in tobacco-producing regions worldwide. It is often responsible for significant economic losses during tobacco production. A rapid, specific, and high-throughput on-site detection method is important for plant disease management. In this study, monoclonal antibody 3H3 and polyclonal antibody 0344 specific for Ralstonia solanacearum were used to prepare a colloidal gold-based immunochromatographic test strip (ITS). Under optimal conditions, the detection limit of the ITS was 105 CFU/mL. The ITS was able to detect different R. solanacearum strains collected from Shandong, Yunnan, Guizhou, and Sichuan provinces in China. Moreover, the ITS was highly specific for R. solanacearum, with no cross-reactivity with Alternaria alternata (Fries) Keissler, Pseudomonas syringae pv. angulata, and P. syringae pv. tabaci. Furthermore, R. solanacearum-spiked tobacco leaves and soil were used to evaluate the matrix interference of the developed ITS, which indicated the test strip was unaffected by leaf size or soil abundance.

Enabling Direct Protein Detection in a Drop of Whole Blood with an "On-Strip" Plasma Separation Unit in a Paper-Based Lateral Flow Strip.

Conventional paper lateral flow assays have low sensitivity and suffer from severe interference from complex human fluid sample matrices, which prevents their practical application in the testing of whole blood samples in the point-of-care settings. To solve this problem, gold nanostar@Raman reporter@silica-sandwiched nanoparticles have been developed as the surface-enhanced Raman scattering (SERS) probes for sensing transduction; and a functionalized filter membrane assembly has been designed and constructed in the paper-based lateral flow strip (PLFS) as a built-in plasma separation unit. In this "on-strip" plasma separation unit, three layers of filter membranes are stacked and surface-modified to maximize the separation efficiency and the plasma yield. As a result, the integrated PLFS has been successfully used for the detection of carcinoembryonic antigen (CEA) in 30 µL of whole blood with the assistance of a portable Raman reader, achieving a limit of detection of 1.0 ng mL-1. In short, this report presents an inexpensive, disposable, portable, and field-deployable paper-based device as a general point-of-care testing tool for protein biomarker detection in a drop of whole blood.

Sequence-Specific Detection of ORF1a, BRAF, and ompW DNA Sequences with Loop Mediated Isothermal Amplification on Lateral Flow Immunoassay Strips Enabled by Molecular Beacons.

Loop-mediated isothermal amplification (LAMP) holds great potential for point-of-care (POC) diagnostics due to its speed and sensitivity. However, differentiation between spurious amplification and amplification of the target sequence is a challenge. Herein, we develop the use of molecular beacon (MB) probes for the sequence-specific detection of LAMP on commercially available lateral flow immunoassay (LFIA) strips. The detection of three unique DNA sequences, including ORF1a from SARS-CoV-2, is demonstrated. In addition, the method is capable of detecting clinically relevant single-nucleotide polymorphisms (BRAF V600E). For all sequences tested, the LFIA method offers similar sensitivity to fluorescence detection using a qPCR instrument. We also demonstrate the coupling of the method with solid-phase microextraction to enable isolation and detection of the target sequences from human plasma, pond water, and artificial saliva. Lastly, a 3D printed device is designed and implemented to prevent contamination caused by opening the reaction containers after LAMP.

Rapid and sensitive recombinase polymerase amplification combined with lateral flow strips for detecting Candida albicans.

Owing to modern lifestyles and increasing amounts of medical intervention, clinical infections caused by conditionally pathogenic fungi are becoming increasingly serious. Among these, Candida albicans is the most common. Therefore, the rapid and accurate detection of this pathogenic fungus is important to guiding the selection of clinical therapeutic agents. Recombinase polymerase amplification (RPA) combined with lateral flow strips (LFS) is a promising molecular detection method with the advantages of rapidity, simplicity of operation and high sensitivity. However, this simplicity brings with it the inherent and non-negligible risk of false-positive signals from primer-dimers. In this study, primer-dependent artifacts were eliminated by using probes in the RPA reaction, introducing specific base substitutions to the primer and probe sequences and analyzing and screening the formation of primer-probe complexes. These measures were rigorously tested for efficacy, leading to the creation of an improved RPA-LFS system. The standardized method enabled the specific detection of C. albicans within 25 min at 37 °C without interference. The system had a detection limit of 1 CFU per reaction without DNA purification or 102 fg genomic DNA/50 µL. The detection sensitivity was not affected by the presence of other fungal DNA. The RPA-LFS method can therefore be used to detect clinical samples, and the results are accurate and consistent in comparison with those obtained using quantitative PCR. This study provides a paradigm for eliminating the risk of false-positive primer dimers in isothermal amplification assays and establishes a simple and easy method for the detection of C. albicans.

Determination of robenidine in shrimp and chicken samples using the indirect competitive enzyme-linked immunosorbent assay and immunochromatographic strip assay.

In this study, the monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic strip assay were developed for the rapid screening of robenidine hydrochloride (ROBH) in samples. The 50% inhibitory concentration (IC50) of ROBH was 0.927 ng mL-1, and the standard curve showed a linear correlation coefficient of 0.99932. There was no cross-reaction between ROBH and other commonly used anticoccidial drugs, which indicated that the monoclonal antibody had high specificity. The recoveries of ic-ELISA were in the range of 87.8% to 102.0%. The immunochromatographic strip assay displayed cut-off values of 10, 5 and 10 ng g-1 for shrimp, chicken breast and chicken liver samples, respectively. In addition, the results can be obtained within 10 min by naked eye observations. And in sample analysis, the results of ic-ELISA and the immunochromatographic strip assay were in accordance with those of LC-MS/MS. Thus, the ic-ELISA and immunochromatographic strip assay are effective methods for the detection of ROBH in shrimp, chicken breast and chicken liver samples.

Solvothermal synthesis of α-Fe2O3 polyhedrons and its application in an immunochromatographic strip test for the detection of foodborne pathogen Listeria monocytogenes.

The immunochromatographic strip test (ICST) is a powerful on-site detection technology due to its unique advantages of simplicity, rapidity, and readability by the naked eye. Here we illustrate the potential of α-Fe2O3 polyhedrons as a novel visual label, which exhibit advantages of high stability and economy, for the detection of Listeria monocytogenes (L. monocytogenes) as a model foodborne pathogen. A low-cost and simple one-step solvothermal approach was developed for the synthesis of α-Fe2O3 polyhedrons; the average diameter of the α-Fe2O3 polyhedrons is about 200 nm. The crystal structure and morphology of α-Fe2O3 polyhedrons were characterized by x-ray diffraction and transmission electron microscope. α-Fe2O3 polyhedrons were immunized with anti-L. monocytogenes antibody to prepare an antibody-colloidal α-Fe2O3 polyhedron ICST. Visual detection can be obtained directly by the naked eye within 10 min. The detection limit of L. monocytogenes by α-Fe2O3 polyhedron ICST assay was 3.8 × 106 and 5.6 × 106 CFU/ml of pure culture and artificially spiked orange juice drink sample, respectively. Results indicated that the antibody-colloidal α-Fe2O3 polyhedron ICST is a rapid, simple, and low-cost assay. This approach showed great potential in the application of foodborne pathogen detection concerning food safety.

A Fluorescent Probe for the Fast Detection of Hypochlorite and its Applications in Water, Test Strip and Living Cells.

Hypochlorite (ClO-) mediated by oxidative stress play an important role in the body's defense system due to their physiological and pathological significance. In this work, a new and simple probe was designed and synthesized to detect hypochlorite. This probe could rapidly respond to hypochlorite in a short time (20 s) in aqueous media, and showed excellent selectivity and sensitivity, and a wide pH range of 3 ̶ 12, as well as the low detection limit of 1.44 nM. In addition, it was successfully applied to the detection of ClO- in water sample, test paper experiment, and cell imaging.

Competitive immunochromatographic test strips for the rapid semi-quantitative analysis of the biologically active bitter glycoside, amarogentin.

Amarogentin (AG), a biologically active secoiridoid glycoside, is responsible for the efficacy of Gentianaceae based medications. Thus, qualitative and quantitative analyses of AG are of significance for batch to batch quality control purposes. By conjugating colloidal gold nanoparticles with the AG-specific monoclonal antibody, MAb 1E9, we were able to develop a single-step competitive immunochromatographic assay (ICA) for simple quantification of the AG content in plant samples. With a limit of detection of 250 ng/mL, the analytical results were obtained after immersing the ICA test strip in the detection mixture for 15 min. This new ICA is superior to conventional ICAs as it is considerably faster due to the speed with which the test strips can be produced and the omission of the time-consuming preparation phase that was previously required to make the fiber pad. Moreover, our ICA only needs a small amount of analyte (20 µL).The reliability of the reported test strip was confirmed by comparing its semi-quantitative results with those obtained via an indirect competitive enzyme-linked immunosorbent assay (icELISA). The positive correlation between these methods (R2 = 0.984) indicated that this new ICA could be applied for the semi-quantitative analysis of the AG content in plant samples.

Combining Electrochemiluminescence Detection with Aptamer-Gated Indicator Releasing Mesoporous Nanoparticles Enables ppt Sensitivity for Strip-Based Rapid Tests.

The combination of electrogenerated chemiluminescence (ECL) and aptamer-gated indicator delivering (gAID) magnetic mesoporous silica nanoparticles embedded into glass fibre paper functionalised with poly(ethyleneglycol) and N-(3-triethoxysilylpropyl)diethanolamine allowed the development of a rapid test that detects penicillin directly in diluted milk down to 50±9 ppt in <5 min. Covalent attachment of the aptamer "cap" to the silica scaffold enabled pore closure through non-covalent electrostatic interactions with surface amino groups, while binding of penicillin led to a folding-up of the aptamer thus releasing the ECL reporter Ru(bpy)32+ previously loaded into the material and letting it be detected after lateral flow by a smartphone camera upon electrochemical excitation with a screen printed electrode inserted into a 3D-printed holder. The approach is simple, generic and presents advantages with respect to sensitivity, measurement uncertainty and robustness compared with conventional fluorescence or electrochemical detection, especially for point-of-need analyses of challenging matrices and analytes at ultra-trace levels.

Disposable Nafion-Coated Single-Walled Carbon Nanotube Test Strip for Electrochemical Quantitative Determination of Acetaminophen in a Finger-Prick Whole Blood Sample.

A disposable electrochemical test strip for the quantitative point-of-care (POC) determination of acetaminophen (paracetamol) in plasma and finger-prick whole blood was fabricated. The industrially scalable dry transfer process of single-walled carbon nanotubes (SWCNTs) and screen printing of silver were combined to produce integrated electrochemical test strips. Nafion coating stabilized the potential of the Ag reference electrode and enabled the selective detection in spiked plasma as well as in whole blood samples. The test strips were able to detect acetaminophen in small 40 µL samples with a detection limit of 0.8 µM and a wide linear range from 1 µM to 2 mM, well within the required clinical range. After a simple 1:1 dilution of plasma and whole blood, a quantitative detection with good recoveries of 79% in plasma and 74% in whole blood was achieved. These results strongly indicate that these electrodes can be used directly to determine the unbound acetaminophen fraction without the need for any additional steps. The developed test strip shows promise as a rapid and simple POC quantitative acetaminophen assay.

Paper-Based Strip for Ultrasensitive Detection of OSCC-Associated Salivary MicroRNA via CRISPR/Cas12a Coupling with IS-Primer Amplification Reaction.

As the most common malignancy in humans, oral squamous cell carcinoma (OSCC) not only harms the people's health but also undermines their confidence after facial surgery. Early detection and treatment can effectively reduce these damages. The unique collateral trans-cleavage nuclease activity of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system was utilized to realize the detection of nucleic acid with high sensitivity. So, in this work, we designed a point-of-care testing (POCT) platform for the detection of OSCC-associated salivary hsa-miRNA 31-5p (miR-31) via the cascade signal amplification of "invading stacking primer" (IS-primer) amplification reaction (ISAR), CRISPR/Cas12a, and dual-mode paper-based strip (dm-Strip). To amplify the detection signal of trace miR-31, the cascade signal amplification of CRISPR/Cas12a system coupling with ISAR was designed in a one-pot reaction at a constant temperature. The target miR-31 could activate the ISAR to generate numerous DNAs, which would further trigger the trans-cleavage effect of Cas12a to catalyze the nonspecific single-stranded DNA (ssDNA) cleavage. This ssDNA was labeled with digoxin and biotin at the 5' and 3' termini (digoxin/ssDNA/biotin), respectively, which led to generate the naked-eye signal and fluorescent signal of the designed dm-Strip. The whole detection time was 90 min with limit-of-detection (LOD) down to aM level. This ISAR/Cas12a-based dm-Strip (ISAR/Cas12a-dmStrip) allowed for the portable and ultrasensitive detection of miRNA, an important step in early diagnosis of OSCC and biomedical research.

Cross-priming isothermal amplification combined with nucleic acid test strips for detection of meat species.

Adulteration of high-quality meat with their cheaper counterparts can be minimized by rapid and reliable methods for detecting meat species. Here an isothermal cross-primer amplification (CPA) technique combined with colloidal gold nucleic acid test strips (CPA strips) was developed to differentiate cow, sheep, arctic fox, and pig meat. A simple primer design for multiplex differentiation using a universal single-labeled CPA primer system and four detection-level species-specific labeling primers were analyzed by colloidal gold-based test strip assay. Moreover, simultaneous detection of fox and pig meat on a double-test line strip was feasible. The CPA strip assay indicated a lower amounts sensitivity of 0.3 ng DNA when one targeted species was tested and a detection limit of 1% when arctic fox meat was detected in the meat mixtures. Using a minimal set of primers, this study provides a promising tool for detecting the species of different types of meat using a constant temperature amplification technology.

Development and application of a colloidal carbon test strip for the detection of antibodies against Mycoplasma bovis.

Mycoplasma bovis (M. bovis) is an important bovine mycoplasma implicated in economically important clinical diseases, such as respiratory diseases, otitis media, and mastitis. The prevalence of M. bovis-associated mastitis in both cattle and buffaloes has been increasingly recognized as a global problem. High morbidity rates and consequential economic losses have been devastating to the affected cattle and buffalo farms, especially those in developing countries. Therefore, a rapid and accurate method is urgently needed to detect M. bovis. In this study, a rapid and simple lateral flow strip for detecting antibodies against M. bovis was established that used carbon nanoparticles (CNPs) as the labelled materials. The results from the test strip were highly consistent with those from ELISA. The test showed high specificity (100%) and no cross-reaction with other bovine pathogens. The detection sensitivity of the test was also relatively high (97.67%). All the results indicated that the colloidal carbon test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for detecting antibodies against M. bovis at cattle farms.

Au@Pd Nanopopcorn and Aptamer Nanoflower Assisted Lateral Flow Strip for Thermal Detection of Exosomes.

Conventional lateral flow biosensing technologies face the dual formidable challenges of poor sensitivity and cumbersome quantitative devices. Here, we developed a Au@Pd nanopopcorn and aptamer nanoflower assisted lateral flow strip (ANAN-LFS) with a thermal signal output to improve detection sensitivity. Moreover, a smartphone-based thermal reader was designed and meticulously optimized to hand-held style, realizing the essential portability of this quantitative device. Experimental studies revealed that the synthesized Au@Pd nanopopcorns clearly red-shifted into the near-infrared region, thus resulting in a higher photothermal response than the standard gold nanoparticles. Aptamer nanoflowers enhanced the system's biorecognition ability significantly compared with single-stranded aptamers due to their functional spatial structure, thus resulting in an even greater improvement in the sensitivity of the ANAN-LFS. With exosomes as model targets, the limit of detection (LOD) was calculated to be 1.4 × 104 exosomes/µL, which exhibited a 71-fold improved analytical performance. The feasibility of this system for detecting spiked biological samples at clinical concentrations was also confirmed. These results suggest that the proposed strategy of integrating a ANAN-LFS with a smartphone-based thermal reader has great potential as a powerful tool for bioanalytical applications, offering the combined unique advantages of high sensitivity and expedient portability.

An HRP-labeled lateral flow immunoassay for rapid simultaneous detection and differentiation of influenza A and B viruses.

Rapid and sensitive diagnosis of influenza is urgently needed to address the limitations of low sensitivity associated with current rapid tests available for clinics and on-site monitoring. A novel horseradish peroxidase (HRP)-labeled lateral flow immunoassay strip (HRP-LFIA) for rapid simultaneous detection and differentiation of influenza A (INF A) and influenza B (INF B) viruses were developed. This immunoassay was based on the signal amplification by the HRP-catalyzed oxidation of 3, 3', 5, 5'-tetramethylbenzidine forming a colored insoluble product, which was proportional to the analyte concentration. Compared with conventional gold-colloidal based strips, an analytical sensitivity enhancement of more than one order of magnitude for thirteen INF virus isolates was observed. A total of 1487 swabs obtained from persons with influenza-like illnesses were tested for the presence of INF A and B viruses using real-time reverse transcription polymerase chain reaction (rRT-PCR) as the reference criterion. The overall sensitivities of HRP-LFIA were 77.5% (100/129) and 71.2% (116/163) for INF A and INF B, respectively. The overall specificities were 99.8% (1144/1146) and 99.8% (918/920), respectively. The nasopharyngeal sampling method yielded higher sensitivity rates of 90.2% (55/61) and 82.6% (71/86). In conclusion, this user-friendly assay could be a promising rapid detection method for rapid screening of INF A and INF B viruses.

Gold nanoparticles of different shape for bicolor lateral flow test.

Spherical gold nanoparticles are the most commonly used marker in lateral flow assays. However, the widespread practice of using identical coloration for the test and control zones of test strips can lead to erroneous interpretations of the assay's results. We propose an immunochromatographic test strip with lines of different colors. For this purpose, gold nanoparticles of different shapes were used, namely blue nanoflowers in the test zone and red gold nanospheres in the control zone. A detailed synthesis procedure for nanoparticles and their conjugates is considered and design parameters for optimal results are described. For the first time, nanoparticles of different shapes have been combined in the test strip with indirect labeling of specific antibodies (via their interaction with labeled secondary antibodies). Using the T-2 toxin (T2T) as an example, an instrumental detection limit of 30 pg/ml and a working range 0.06-0.9 ng/mL were achieved in an analysis of water-organic corn extracts.

Strip-shaped Co3O4 as a peroxidase mimic in a signal-amplified impedimetric zearalenone immunoassay.

An impedimetric immunoassay was designed for ultrasensitive determination of zearalenone (ZEN). It is making use of the peroxidase-like activity of strip-shaped Co3O4 (ssCo3O4) which catalyzes the oxidation of 4-chloro-1-naphthol to produce an insoluble precipitate in the presence of H2O2. The precipitate is electrically nonconductive and accumulates on the electrode, thereby retarding the electron transfer from the redox probe ferro/ferricyanide to the surface of electrode. This amplifies the impedimetric signal in accordance with logarithm of the concentration of ZEN. The electrode was further modified with TiO2 mesocrystals (TiO2 MCs) which improve the capture of more analytes and increase the performance of the immunoassay. Under optimized experimental condition, the impedimetric signal increased linearly with the logarithm of the ZEN concentration range between 0.1 fg·mL-1 to 10 pg·mL-1. The detection limit is of 33 ag· mL-1. Graphical abstractThis work describes an impedimetric immunoassay based on the use of strip-shaped Co3O4 that catalyzes the production of an insoluble precipitate in the presence of H2O2 on the surface of a glassy carbon electrode. The effect was used for signal amplification in an electrochemical immunoassay for zearalenone.

Development of A Lateral Flow Highway: Ultra-Rapid Multitracking Immunosensor for Cardiac Markers.

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic "tracks" for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1-1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%-15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.

Aptamer-based fluorometric lateral flow assay for creatine kinase MB.

A group of aptamers possessing high specificity and affinity for creatine kinase MB (CKMB) was obtained by magnetic systematic evolution of ligands by exponential enrichment. Two aptamers (referred to as C.Apt.21 and C.Apt.30) were found to possess adequately low Kd values. They form a well suited pair for CKMB binding. By using fluorescent microspheres, an aptamer-based lateral flow assay was developed. It is portable, economical, and sensitive. The limit of detection for CKMB is as low as 0.63 ng·mL-1, and the assay works in the 0.005 - 2 µg·mL-1 CKMB concentration range. The method is specific for CKMB, and biomarkers for AMI (such as cardiac troponin I and myoglobin) and serum do not interfere. The strip is highly accurate as shown by analysis of spiked serum samples which gave recoveries ranging between 88 and 117%. Graphical Abstract Schematic of the test strip and sandwich aptamer-based fluorometric lateral flow assay for creatine kinease. The detection is based on the specific affinity between CKMB and selected aptamers to form a sandwich structure.

A test strip for ochratoxin A based on the use of aptamer-modified fluorescence upconversion nanoparticles.

An aptamer-based test strip is described for visual and instrumental determination of the mycotoxin ochratoxin A (OTA). It is based on the use of NaYF4:Yb,Er upconversion nanoparticles (UCNPs) as a label for the aptamer and on the competition between OTA and its complementary sequence for an OTA-specific aptamer. To improve the analytical performance, the optical properties of the UCNPs, the fluidity of the UCNP-aptamer conjugate, and the migration rate on the nitrocellulose membranes were investigated. Under the optimal experimental conditions and by using a 980-nm laser, the relative fluorescence intensity (test line value/control line value) is proportional to the logarithm of the OTA concentration over a range from 5 to 100 ng·mL-1 (R2 = 0.9955). The limit of the detection is 1.86 ng·mL-1. This aptamer based flow assay can be performed within 15 min and has no serious cross-sensitivity to potentially interfering species. It was successfully applied to the determination of OTA in spiked wheat and beer samples. Graphical abstract An aptamer-based upconversion fluorescent strip based on the use of NaYF4:Yb,Er nanoparticles was developed for sensitive detection of Ochratoxin A. The limit of the detection was determined as 1.86 ng·mL-1. The assay can be performed within 15 min, indicating its great potential in point-of-care testing.