A sensitive intelligent point-of-care test method for tert-butylhydroquinone in edible oil via a test strip with a smartphone.

Tert-butylhydroquinone (TBHQ) is easily overused or illegally added to edible oil and attracts a growing concern because of its cytotoxic, liver-damaging, and carcinogenic effects. Thus, a sensitive and intelligent point-of-care testing (iPOCT) method is developed to fulfill the on-site monitoring. This iPOCT method depended on a fluorescent immunochromatographic assay within 15 min. Under optimization, the limit of quantification (LOQ) was calculated as 0.03 µg mL-1. The iPOCT method provided a low limit of detection (LOD) of 0.02 µg mL-1, a wide linear range of 0.03-100 µg mL-1, and great selectivity. Recoveries by the spiking experiments ranged from 97.4% to 103.5% with relative standard deviations (RSDs) of 2.4%-4.9% in soybean, peanut, rapeseed, and corn oil samples. The results showed that the iPOCT method is highly consistent with the high-performance liquid chromatography (HPLC) method.

Simultaneous detection of two subtypes of extracellular vesicles using ultrabright fluorescent nanosphere-based test strips.

In recent years, the cargo profiles of extracellular vesicles (EVs), which were inherited from their parent cells, have emerged as a reliable biomarker for liquid biopsy (LB) in disease diagnosis, prognosis, and treatment monitoring. EVs secreted by different cells exhibit distinct characteristics, particularly in terms of disease diagnosis and prediction. However, currently available techniques for the quantitative analysis of EV cargoes, including enzyme-linked immunosorbent assay (ELISA), cannot specifically identify the cellular origin of EVs, thus seriously affecting the accuracy of EV-based liquid biopsy. In light of this, we here developed ultrabright fluorescent nanosphere (FNs)-based test strips which have the unique capability to specifically assess the levels of PD-L1-positive EVs (PD-L1+ EVs) derived from both tumor cells and immune cells in bodily fluids. The levels of PD-L1+ EV subpopulations in human saliva were quantified using the ultrabright fluorescent nanosphere-based test strips with more convenience and higher efficiency (detection time <30 min). Results demonstrated that the fluorescence intensity of the test line exhibited a good linear relationship respectively with the PD-L1 levels of tumor cell- (R2 = 0.993) and immune cell-derived EVs (R2 = 0.982) in human saliva. By assessing the levels of PD-L1+ EV subpopulations, our test strips hold immense potential for advancing the application of PD-L1+ EV subpopulation-based predictions in tumor diagnosis and prognosis evaluation. In summary, by integrating the benefits of FNs and lateral flow chromatography, we here provide a strategy to accurately measure the cargo levels of EVs originating from diverse cell sources in bodily fluids.

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.

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.

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.

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.

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.

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.

Rapid and quantitative detection of urinary Cyfra21-1 using fluorescent nanosphere-based immunochromatographic test strip for diagnosis and prognostic monitoring of bladder cancer.

Bladder cancer is a common malignant tumour with high recurrence rate. Cytokeratin 19 fragments (Cyfra21-1) in urine has been regarded as a promising biomarker for the prognosis and diagnosis of bladder cancer due to the relevance of its high urinary level to the bladder cancer patients. However, currently detection methods of Cyfra21-1 have their limits, such as complicated steps, limited sensitivity or unsatisfying specificity. In this study, we developed a novel time-resolved fluoroimmuno test strip by using europium chelate microparticle (Eu-CM). Detection was performed in simple steps by carrying drops of sample into the well of the test strip, waiting for 15 min and inserting the strip into a fluorescence strip reader for quantitation. The standard curve equation of the test strip was y = 0.0177x + 0.01 (R2 = .9993). In the analysis of human urine samples (n = 115), it demonstrated a good performance (accuracy: CV < 10%, AUC: 0.989). With the cut-off value of 81 ng/mL, the sensitivity and specificity for bladder cancer were 92.86 and 100%, respectively. In comparison to ELISA and electrochemiluminescence methods, the Eu-CM based time-resolved fluoroimmuno test strip provided a rapid, sensitive and reliable method for monitoring bladder cancer. It may be applied as a non-invasive approach for in point-of-care for bladder cancer detection.

[Feasibility analysis of pepsin strip test in diagnosis of laryngopharyngeal reflux disease].

Objective: To evaluate the feasibility of pepsin strip test in the diagnosis of laryngopharyngeal reflux. Methods: From August 2017 to September 2018,80 patients in Department of Otorhniolaryngology Head and Neck Surgery,Chinese People's Liberation Army General Hospital-Six Medical Centre, underwent pepsin strip test and 24-hour multichannel intraluminal impedance(MII)-pH monitoring. The results of the two methods were analyzed for consistency,and 24-hour MII-pH monitoring was used as a statistical reference for the sensitivity and specificity of pepsin strip test in the diagnosis of laryngopharyngeal reflux. Data were analyzed by SPSS 19.0 software. Results: There were 57 patients with positive pepsin test strip and 23 patients with negative pepsin test strip. The score of reflux symptoms and signs, and the positive rate of laryngopharyngeal reflux events in patients with positive pepsin strip test were significantly higher than those in patients with negative pepsin test strip. If there was one or more throat reflux events (including acid reflux,weak acid reflux and alkali reflux) as the positive results of 24-hour MII-pH monitoring,the consistency between the results of pepsin strip and 24-hour MII-pH was moderate (Kappa=0.614). The sensitivity and specificity of pepsin strip were 86.9% (53/61) and 78.9% (15/19) respectively. Conclusions: Pepsin strip detection has the advantages of non-invasive,cheap and easy to operate.As an objective method for early diagnosis of laryngopharyngeal reflux, pepsin strip detectionis feasible,but can not be the final diagnosis for laryngopharyngeal reflux disease.

A turn-on competitive immunochromatographic strips integrated with quantum dots and gold nano-stars for cadmium ion detection.

Immunochromatographic strips (ICSs) are inexpensive, simple, portable, and robust, and therefore have many uses in the medicinal, agricultural, and environmental industries. For detection of small molecules, current ICSs are competitive format (competitive ICSs, CICSs), which only offer a turn-off readout mode, and therefore lead to low sensitivity when evaluating results by the naked eye. To overcome this problem, we report a turn-on CICSs that relies on the ability of gold nano-stars (AuNSs) quenching the signal of quantum dots (QDs). This turn-on CICSs device was applied to detect cadmium ions (Cd2+). The linear detection range (LDR) of the turn-on CICSs was 0.25ng/mL-8ng/mL, and the detection of limit (LOD) was 0.18ng/mL. Compared with traditional turn-off CICSs, the sensitivity of the turn-on CICSs was enhanced by 32 times. The turn-on CICSs also has a high specificity and high recovery for the detection of Cd2+ in Pearl River (95-112%) and tap water samples (103.5-116.67%). Therefore, we believe the turn-on CICSs offers great potential for the detection of other small molecules in clinical diagnostics, food safety investigations, and environment pollution monitoring.

A simple and cost effective method for preparing FL and LG solutions.

PURPOSE: The purpose of this study was to develop a clinically feasible method for obtaining dye concentrations of 2% fluorescein (FL) and 1% lissamine green (LG) by soaking commercially available dye impregnated strips in saline. METHODS: Calibration curves were established to related known concentrations of dye to prepared FL fluorescence and LG absorbance. To determine the optimum number of dye strips and soaking times (preliminary testing), 1, 2, 3 FL or LG strips were soaked in 200 µl commercially available saline for 0.5, 1, 2, 3, 4 and 5 min, using calibration curves to determine FL and LG concentrations. The best combination of number of dye strips and soaking time was soaking 3FL and 3LG strips for 5 min and these were finally tested in 2 ml centrifuge tubes, selected for ease of use in a clinical setting. RESULTS: Preliminary testing indicated that soaking 3 FL or 3 LG strips for 5 min in saline yielded an average (±standard deviation) of 2.0 ± 0.000% FL and 0.93 ± 0.010% LG. Final testing of FL in centrifuge tubes (strips soaked for 3-15 min) yielded an average of 1.99 ± 0.040% FL, with no significant difference among time periods or dye lots tested. However, LG showed more variable results with an average of 0.80 ± 0.160% LG (5-15 min), with significant differences among dye lots and times (2-way ANOVA, p < 0.05). CONCLUSIONS: This simple, reliable and relatively inexpensive method involves soaking 3 FL or LG strips in saline solution, yielding concentrations close to the 2%FL and 1%LG recommended for clinical trials, although LG showed more variability.

Development of a lateral-flow immunochromatographic strip using gold nanoparticles for Helicobacter pylori detection.

Helicobacter pylori (H. pylori) plays an important role in the development of chronic gastritis, peptic ulcer diseases, gastric adenocarcinoma, and gastric mucosal associated lymphoid tissue (MALT) lymphoma. The standard methods of bacterial staining, bacterial culture, and urease testfor the detection of H. pylori are time consuming and invasive. Non-invasive testing plays a significant role in the test-and-treat approach to H. pylori management. Lateral flow immunoassay (LFIA) is a promising method for pathogenic detection that is fast, easy to use, and low cost. In the present study, the authors developed an H. pylori LFIA strip using gold nanoparticles for H. pylori detection. The results reported that 20 µg of anti-H. pylori antibody mixed with 1 mL AuNPs solution and incubated for 2 hours was the best concentration preparation for application coverage over the gold nanoparticle surface. The limit of detection observable by the naked eye was 15 µg of H. pylori protein lysate. The findings of this study suggest the possible future development of an H. pylori LFLA strip for fast, easy to use, and low-cost diagnostic testing.

Filter Paper-based Nucleic Acid Storage in High-throughput Solid Tumor Genotyping.

Molecular testing of tumors from formalin-fixed paraffin-embedded (FFPE) tissue blocks is central to clinical practice; however, it requires histology support and increases test turnaround time. Prospective fresh frozen tissue collection requires special handling, additional storage space, and may not be feasible for small specimens. Filter paper-based collection of tumor DNA reduces the need for histology support, requires little storage space, and preserves high-quality nucleic acid. We investigated the performance of tumor smears on filter paper in solid tumor genotyping, as compared with paired FFPE samples. Whatman FTA Micro Card (FTA preps) smears were prepared from 21 fresh tumor samples. A corresponding cytology smear was used to assess tumor cellularity and necrosis. DNA was isolated from FTA preps and FFPE core samples using automated methods and quantified using SYBR green dsDNA detection. Samples were genotyped for 471 mutations on a mass spectrophotometry-based platform (Sequenom). DNA concentrations from FTA preps and FFPE correlated for untreated carcinomas but not for mesenchymal tumors (Spearman σ=0.39 and σ=-0.1, respectively). Average DNA concentrations were lower from FTA preps as compared with FFPE, but DNA quality was higher with less fragmentation. Seventy-six percent of FTA preps and 86% of FFPE samples generated adequate DNA for genotyping. FTA preps tended to perform poorly for collection of DNA from pretreated carcinomas and mesenchymal neoplasms. Of the 16 paired DNA samples that were genotyped, 15 (94%) gave entirely concordant results. Filter paper-based sample preservation is a feasible alternative to FFPE for use in automated, high-throughput genotyping of carcinomas.

A visual strip sensor for determination of iron.

A visual strip has been developed for sensing iron in different aqueous samples like natural water and fruit juices. The sensor has been synthesized by UV-radiation induced graft polymerization of acrylamide monomer in microporous poly(propylene) base. For physical immobilization of iron selective reagent, the in situ polymerization of acrylamide has been carried out in the presence of 1,10-phenanthroline. The loaded strip on interaction with Fe(II) in aqueous solution turned into orange red color and the intensity of the color was found to be directly proportional to the amount of Fe(II) in the aqueous sample. The minimal sensor response with naked eye was found for 50ngmL(-1) of Fe in 15min of interaction. However, as low as 20ngmL(-1) Fe could be quantified using a spectrophotometer. The detection limit calculated using the 3s/S criteria, where 's' is the standard deviation of the absorbance of blank reagent loaded strip and 'S' is the slope of the linear calibration plot, was 1.0ngmL(-1). The strip was applied to measure Fe in a variety of samples such as ground water and fruit juices.

A smartphone platform for the quantification of vitamin D levels.

Vitamin D deficiency has been linked to a number of diseases and adverse outcomes including: osteoporosis, infections, diabetes, cardiovascular diseases, and even cancer. At present the vast majority of vitamin D testing is performed in large-scale laboratories at the request of a physician as part of an annual panel of blood tests. Here we present a system for rapid quantification of vitamin D levels on a smartphone. The system consists of a smartphone accessory, an app, and a test strip that allows the colorimetric detection of 25-hydroxyvitamin D using a novel gold nanoparticle-based immunoassay. We show that the system can be used to accurately measure physiological levels of 25-hydroxyvitamin D with accuracy better than 15 nM and a precision of 10 nM. We compare our system with well-established ELISA test kits for serum samples of unknown concentration and demonstrate equivalency of the results. We envision this as the first step towards the development of the NutriPhone, a comprehensive system for the analysis of multiple vitamins and micronutrients on a smartphone.

Eco-friendly plasmonic sensors: using the photothermal effect to prepare metal nanoparticle-containing test papers for highly sensitive colorimetric detection.

Convenient, rapid, and accurate detection of chemical and biomolecules would be a great benefit to medical, pharmaceutical, and environmental sciences. Many chemical and biosensors based on metal nanoparticles (NPs) have been developed. However, as a result of the inconvenience and complexity of most of the current preparation techniques, surface plasmon-based test papers are not as common as, for example, litmus paper, which finds daily use. In this paper, we propose a convenient and practical technique, based on the photothermal effect, to fabricate the plasmonic test paper. This technique is superior to other reported methods for its rapid fabrication time (a few seconds), large-area throughput, selectivity in the positioning of the NPs, and the capability of preparing NP arrays in high density on various paper substrates. In addition to their low cost, portability, flexibility, and biodegradability, plasmonic test paper can be burned after detecting contagious biomolecules, making them safe and eco-friendly.

Development of a colloidal gold-based immunochromatographic test strip for screening of microalbuminuria.

A rapid immunochromatography (ICG) assay based on antibody colloidal gold nanoparticles specific to human serum albumin (HSA) was developed, and its applications for primary screening of HSA in the urine were evaluated. A monoclonal antibody (MAb) specific to HSA was produced from cloned hybridoma cells (EMRC1) and used to develop an ICG strip. The nanocolloidal gold, with an average particle diameter of 20 nm, was synthesized and labeled MAb as the detection reagent. An antibody colloidal gold probe was applied on the conjugate pad, and HSA antigen was immobilized to a nitrocellulose membrane as the capture reagent to prepare the ICG strip test. This test required only 10 min to accomplish a semiquantitative detection of albumin. The sensitivity to urinary albumin was found to be approximately 20 µg/mL, and the analytical range was 20-25 µg/mL. The reliability of the testing procedures was examined by carrying out the ICG strip test with 40 urine samples and comparing the results of these tests with those obtained via immunoturbidimetry. The ICG strip was adequately sensitive and accurate for a rapid screening of HSA in the urine.

Glycidyl methacrylate-co-N-vinyl-2-pyrrolidone coated polypropylene strips: synthesis, characterization and standardization for dot-enzyme linked immunosorbent assay.

Glycidyl methacrylate and N-vinyl-2-pyrrolidone (GMA-co-NVP) copolymers with various GMA:NVP ratios were synthesized by solution polymerization technique in toluene using 2,2'-azobisisobutyronitrile (AIBN) as free radical initiator and dip coated onto polypropylene strips. The copolymer composition in polymeric coatings was confirmed by proton NMR spectroscopy. Various techniques like FTIR, SEM and contact angle were used for surface characterization of the polymer coatings. These polymer coated strips were evaluated and standardized for their application in dot-ELISA in two steps. In first step, specificity, sensitivity and reproducibility of the assay on developed polymer coated strips was evaluated through a model system using rabbit anti-goat IgG, goat anti-rabbit IgG and goat anti-rabbit IgG HRP (horseradish peroxidase)-conjugate. Polymer coating with GMA-NVP mol% ratio of 78:22 was able to detect rabbit anti-goat IgG antibody at a concentration as low as 2 ng mL(-1) with 1% BSA as blocking agent using antispecies IgG peroxidase conjugate diluted 1500 times. In the second step, the sensitivity and specificity of the developed system was established with human blood and finally used to identify the source of mosquito blood meal which is an important parameter in epidemiological studies, particularly in determining the role of mosquito in malaria transmission. The time duration of standardized assay with developed polymer coated strips was cut down to one hour compared to the 3-4h required in usual dot-ELISA.

Paper-based bioassays using gold nanoparticle colorimetric probes.

The majority of bioassays utilize thermosensitive reagents (e.g., biomolecules) and laboratory conditions for analysis. The developing world, however, requires inexpensive, simple-to-perform tests that do not require refrigeration or access to highly trained technicians. To address this need, paper-based bioassays using gold nanoparticle (AuNP) colorimetric probes have been developed. In the two prototype DNase I and adenosine-sensing assays, blue (or black)-colored DNA-cross-linked AuNP aggregates were spotted on paper substrates. The addition of target DNase I (or adenosine) solution dissociated the gold aggregates into dispersed AuNPs, which generated an intense red color on paper within one minute. Both hydrophobic and (poly(vinyl alcohol)-coated) hydrophilic paper substrates were suitable for this biosensing platform; by contrast, uncoated hydrophilic paper caused "bleeding" and premature cessation of the assay due to surface drying. The assays are surprisingly thermally stable. During preparation, AuNP aggregate-coated papers can be dried at elevated temperatures (e.g., 90 degrees C) without significant loss of biosensing performance, which suggests the paper substrate protects AuNP aggregate probes from external nonspecific stimuli (e.g., heat). Moreover, the dried AuNP aggregate-coated papers can be stored for at least several weeks without loss of the biosensing function. The combination of paper substrates and AuNP colorimetric probes makes the final products inexpensive, low-volume, portable, disposable, and easy-to-use. We believe this simple, practical bioassay platform will be of interest for use in areas such as disease diagnostics, pathogen detection, and quality monitoring of food and water.