Europium (III) chelate microparticle-based lateral flow immunoassay strips for rapid and quantitative detection of antibody to hepatitis B core antigen.
ABSTRACT: Quantitative hepatitis B core antigen (anti-HBc) measurements could play an important role in evaluating therapeutic outcomes and optimizing the antiviral therapy of chronic hepatitis B infection. In this study, we have developed a simple and rapid fluorescence point-of-care test based on a lateral flow immunoassay (LFIA) method integrated with Eu (III) chelate microparticles to quantitatively determine anti-HBc concentrations in serum. This assay is based on a direct competitive immunoassay performed on lateral flow test strips with an assay time of 15?min. The Eu (III) chelate microparticle-based LFIA assay could quantitatively detect anti-HBc levels with a limit of detection of 0.31 IU mL-1, and exhibited a wide linear range (0.63-640 IU mL-1). The intra- and inter-assay coefficients of variation for anti-HBc were both less than 10% and a satisfactory dilution test and accuracy were demonstrated. There were no statistically significant differences in sensitivity or specificity in serum samples between the Eu (III) chelate microparticle-based LFIA strips and the Abbott Architect kit. A simple, rapid and effective quantitative detection of anti-HBc was possible using the Eu (III) chelate microparticle-based LFIA strips. The strips will provide diagnostic value for clinical application.
Project description:Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, is the predominant cause of severe enteropathogenic diarrhea in swine. A simple, rapid, specific, and sensitive method is critical for monitoring PEDV on pig farms. In this study, a simple and rapid lateral flow immunoassay detection system that integrates europium (Eu) (III) chelate microparticles was developed to identify PEDV in fecal swabs. This newly developed diagnostic sandwich immunoassay utilizes lateral flow test strips (LFTSs). The fluorescence peak heights of the test line (HT) and the control line (HC) were measured using a fluorescence strip reader, and the HT/HC ratio was used for quantitation. The limit of detection of PEDV with this LFTS was ??ten times the median tissue culture infectious dose (TCID50) per mL??. Fecal swab samples were used to determine the cutoff value. Field samples, various PEDV strains and other viruses were used to determine the sensitivity and specificity of the Eu (III) chelate microparticle-based LFTSs, which were 97.8% and 100%, respectively, with a cutoff value of 0.05, as compared with reverse transcription polymerase chain reaction (RT-PCR). In samples from piglets experimentally infected with PEDV, the results were in high agreement with those obtained by RT-PCR. Epidemiological surveillance of PEDV using the LFTSs ??in areas threatened by African swine fever virus?? suggested that the PEDV positive rate on pig farms had significantly decreased, mainly due to the implementation of strict biosecurity measures. The results indicate that the Eu (III) chelate microparticle-based LFTS system is a rapid, sensitive, and reliable method for the identification of PEDV, indicating its suitability for epidemiological surveillance of PEDV infection.
Project description:Lateral flow immunoassay (LFIA) is a convenient tool for rapid field-based control of various bacterial targets. However, for many applications, the detection limits obtained by LFIA are not sufficient. In this paper, we propose enlarging gold nanoparticles' (GNPs) size to develop a sensitive lateral flow immunoassay to detect Ralstonia solanacearum. This bacterium is a quarantine organism that causes potato brown rot. We fabricated lateral flow test strips using gold nanoparticles (17.4 ± 1.0 nm) as a label and their conjugates with antibodies specific to R. solanacearum. We proposed a signal enhancement in the test strips' test zone due to the tetrachloroauric (III) anion reduction on the GNP surface, and the increase in size of the gold nanoparticles on the test strips was approximately up to 100 nm, as confirmed by scanning electron microscopy. Overall, the gold enhancement approach decreased the detection limit of R. solanacearum by 33 times, to as low as 3 × 10? cells?mL?1 in the potato tuber extract. The achieved detection limit allows the diagnosis of latent infection in potato tubers. The developed approach based on gold enhancement does not complicate analyses and requires only 3 min. The developed assay together with the sample preparation and gold enlargement requires 15 min. Thus, the developed approach is promising for the development of lateral flow test strips and their subsequent introduction into diagnostic practice.
Project description:Escherichia coli O157:H7 is regarded as one of the most harmful pathogenic microorganisms related to foodborne diseases. This paper proposes a rapid-detection biosensor for the sensitive and quantitative analysis of E. coli O157:H7 in biological samples by surface-enhanced Raman scattering (SERS)-based lateral flow immunoassay (LFIA). A novel gold-shell silica-core (SiO2/Au) nanosphere (NP) with monodispersity, good stability, and excellent SERS activity was utilized to prepare high-performance tags for the SERS-based LFIA system. The SiO2/Au SERS tags, which were modified with two layers of Raman reporter molecules and monoclonal antibodies, effectively bind with E. coli O157:H7 and form sandwich immune complexes on the test lines. E. coli O157:H7 was quantitatively detected easily by detecting the Raman intensity of the test lines. Under optimal conditions, the limit of detection (LOD) of the SiO2/Au-based SERS-LIFA strips for the target bacteria was 50 cells/mL in PBS solution, indicating these strips are 2,000 times more sensitive than colloidal Au-based LFIA strips. Moreover, the proposed assay demonstrated high applicability in E. coli O157:H7 detection in biological samples, including tap water, milk, human urine, lettuce extract and beef, with a low LOD of 100 cells/mL. Results indicate that the proposed SERS-based LFIA strip is applicable for the sensitive and quantitative determination of E. coli O157:H7.
Project description:Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.
Project description:The crystal structure of title compound, [Eu(NO(3))(3)(C(15)H(15)NO(2))(2)], contains two Schiff base 6-meth-oxy-2-[(4-methyl-phen-yl)imino-meth-yl]phenolate (L) ligands and three independent nitrate ions that chelate to the europium(III) ion via the O atoms. The coordination number of the Eu(III) ion is ten. The L ligands chelate with a strong Eu-O(deprotonated phenolate) bond and a weak Eu-O(meth-oxy) contact, the latter can be inter-preted as the apices of the bicapped square-anti-prismatic Eu(III) polyhedron. Intra-molecular N-H?O hydrogen bonds occur.
Project description:<i>Escherichia coli</i> O157:H7 is known to cause serious diseases including hemorrhagic colitis and hemolytic uremic syndrome. A gold nanoparticle lateral flow immunoassay (Au-LFIA) was used to detect <i>Escherichia coli</i> O157:H7 in ground pork samples. False-positive results were detected using Au-LFIA; a <i>Citrobacter</i><i>freundii</i> strain was isolated from the ground pork samples and identified by using CHROmagar<sup>TM</sup> plates, API 20E, and 16S RNA sequencing. Since <i>C.</i><i>freundii</i> showed cross-reactivity with <i>E. coli</i> O157:H7 when Au-LFIA test strips were used, a novel method combining modified enrichment with a lateral flow immunoassay for accurate and convenient detection of <i>E. coli</i> O157:H7 in ground pork was developed in this study to minimize these false positives. MacConkey broth was optimized for <i>E. coli</i> O157:H7 enrichment and <i>C.</i><i>freundii</i> inhibition by the addition of 5 mg/L potassium tellurite and 0.10 mg/L cefixime. Using the proposed modified enrichment procedure, the false-positive rate of ground pork samples spiked with 100 CFU/g <i>C.</i><i>freundii</i> decreased to 5%.
Project description:<h4>Background</h4>Blood prostate-specific antigen (PSA) levels are widely used as diagnostic biomarkers for prostate cancer. Lateral-flow immunoassay (LFIA)-based PSA detection can overcome the limitations associated with other methods. LFIAbased PSA detection in clinical samples enables prognosis and early diagnosis owing to the use of high-performance signal reporters.<h4>Results</h4>Here, a semiquantitative LFIA platform for PSA detection in blood was developed using Au-Ag nanoparticles (NPs) assembled on silica NPs (SiO2@Au-Ag NPs) that served as signal reporters. Synthesized SiO2@Au-Ag NPs exhibited a high absorbance at a wide wavelength range (400-800 nm), with a high scattering on nitrocellulose membrane test strips. In LFIA, the color intensity of the test line on the test strip differed depending on the PSA concentration (0.30-10.00 ng/mL), and bands for the test line on the test strip could be used as a standard. When clinical samples were assessed using this LFIA, a visual test line with particular color intensity observed on the test strip enabled the early diagnosis and prognosis of patients with prostate cancer based on PSA detection. In addition, the relative standard deviation of reproducibility was 1.41%, indicating high reproducibility, and the signal reporter showed good stability for 10 days.<h4>Conclusion</h4>These characteristics of the signal reporter demonstrated the reliability of the LFIA platform for PSA detection, suggesting potential applications in clinical sample analysis.
Project description:Prevalence and risk factors for isolated antibody to hepatitis B core antigen (anti-HBc) and occult hepatitis B virus (HBV) infection are not well known in human immunodeficiency virus type 1 (HIV-1)-infected pregnant women. It is unclear if women with occult infections are at risk of transmitting HBV to their infants.HIV-1-infected and HBV surface antigen (HBsAg)-negative pregnant women were tested for antibody to HBsAg (anti-HBs) and anti-HBc using enzyme immunoassay. Women with isolated anti-HBc were assessed for occult HBV infection, defined as HBV DNA levels >15 IU/mL, using the Abbott RealTime HBV DNA assay. Infants born to women with isolated anti-HBc and detectable HBV DNA were tested at 4 months of age for HBV DNA. Logistic regression analysis was used to identify factors associated with isolated anti-HBc and occult HBV infection.Among 1812 HIV-infected pregnant women, 1682 were HBsAg negative. Fourteen percent (95% confidence interval [CI], 12%-15%) of HBsAg-negative women had an isolated anti-HBc that was independently associated with low CD4 count, age >35 years, birth in northern Thailand, and positive anti-hepatitis C virus serology. Occult HBV infection was identified in 24% (95% CI, 18%-30%) of women with isolated anti-HBc, representing 2.6% (95% CI, 1.9%-3.5%) of HIV-1-infected pregnant women, and was inversely associated with HIV RNA levels. None of the women with isolated anti-HBc and occult HBV infection transmitted HBV to their infants.HIV-1-infected pregnant women with isolated anti-HBc and occult HBV infection have very low HBV DNA levels and are thus at very low risk to transmit HBV to their infants.
Project description:Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader's analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2?750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.
Project description:The synthesis, characterization, and photophysical properties are reported for several Ln(III) complexes of a tetradentate chelate, 5LIO-MAM, derived from the common flavor enhancer "maltol". Eu(III), Yb(III), and Nd(III) form stable ML2 complexes in aqueous solution that emit in the red or near-infrared (NIR) upon excitation at ca. 330 nm. The synthesis, aqueous stability, and photophysical properties are reported for a novel tetradentate ligand derived from maltol, a commonly used flavor enhancer. In aqueous solution, this chelate forms stable complexes with Ln(III) cations, and sensitized emission was observed from Eu(III), Yb(III), and Nd(III). A comparison with recently reported and structurally analogous ligands reveals a slightly higher basicity but lower complex stability with Eu(III) [pEu = 14.7(1)]. A very poor metal-centered quantum yield with Eu(III) was observed (Phi(tot) = 0.04%), which can be rationalized by the similar energy of the ligand triplet state and the Eu(III) (5)D0 emissive level. Instead, sensitized emission from the Yb(III) and Nd(III) cations was observed, which emit in the NIR.