Development of an immunochromatographic test strip for the rapid detection of aristolochic acid A in herbal medicinal materials.

INTRODUCTION: Plants containing aristolochic acid and its derivatives are nephrotoxic, mutagenic, and carcinogenic to humans; chronic diet poisoning caused by the aristolochic acid is the cause of endemic (Balkan) nephropathy and related cancers. OBJECTIVE: To develop a colloidal gold immunochromatographic test strip (ICS) based on the competitive format for the rapid detection of aristolochic acid A (AA-A) in herbal medicinal materials. MATERIALS AND METHODS: For the ICS based on gold nanoparticles (AuNPs), the antigen [AA-A-bovine serum albumin (BSA)], and goat anti-mouse IgG were drawn on the nitrocellulose membrane as the test line (T line) and the control line (C line), respectively. Monoclonal antibody (MAb)-AuNP conjugates were sprayed onto the conjugate pad. The sensitivity of the ICS was 6 ng/mL, and the test was completed in 10 min. The analysis of AA-A in traditional Chinese medicine samples showed that the ICS results were in good agreement with those obtained by high-performance liquid chromatography methods. CONCLUSION: These results demonstrated that the ICS test could be used as a reliable, rapid, cost-effective, and convenient qualitative tool for on-site screening techniques to detect AA-A in herbal medicinal materials without any special instrumentation.

The colloidal gold nanoparticle-based lateral flow immunoassay for fast and simple detection of plant-derived doping agent, higenamine.

Higenamine (HM), an alkaloid found in various plant species, is obtained when norcoclaurine synthase selectively condenses dopamine and 4-hydroxyphenylacetaldehyde to give (S)-higenamine ((S)-HM). The World Anti-doping Agency has listed HM as a prohibited agent in athletics. As a result, many commercial, academic, and regulatory bodies across the globe are invested in finding a rapid method for (S)-HM detection. In the current study, a lateral flow immunoassay (LFA) was developed in which the relevant biosensor was generated as a conjugate of the monoclonal antibody against (S)-HM (namely, MAb E8) and colloidal gold nanoparticles. The HM-γ-globulin conjugates and rabbit anti-mouse IgG antibodies were placed in the test and control zones, respectively. The free (S)-HM molecules in the samples and the immobilized HM-γ-globulin conjugates competitively reacted with the developed biosensor in the LFA. An inverse relationship existed between the biosensors' visible response, which was noted by the variation in the intensity of a pinkish spot in the test zone, and the content of the free (S)-HM. The limit of detection of the developed LFA was 156 ng/mL. Various validation methods confirmed that the LFA exhibited sufficient sensitivity, selectivity, repeatability, and reliability, making it ideal for (S)-HM detection in plant samples and plant-containing products. The developed system required only a small sample volume (20 µL) and a concise sample preparation time compared with conventional LFAs. Thus, the LFA reported in this study could serve as a rapid response kit for the detection of (S)-HM in plant samples.

An integrated strategy for rapid on-site screening and determination of prometryn residues in herbs.

We produced a prometryn-specific monoclonal antibody and propose a strategy for convenient on-site detection of prometryn residues in herbs for the first time. This strategy has perfect applicability in a complex herbal medicine matrix. The strategy combines a semiquantitative immunochromatographic strip assay with a heterologous indirect competitive ELISA. When there was no matrix interference, the ELISA had a half-maximal inhibitory concentration of 2.6 ng·mL-1 and a limit of detection of 0.2 ng·mL-1. The immunochromatographic strip assay can be completed within 5 min with a visual limit of detection of 1 ng·mL-1. Although the sample matrix had different effects on the sensitivity of the antibody, excellent repeatability and accuracy were achieved. The method was successfully applied for the screening and determination of prometryn residue in multiple complex herb samples for the first time, and the results were in good agreement with those obtained by liquid chromatography-tandem mass spectrometry. The proposed strategy is rapid, of high-throughput, and of low cost, and may be a promising choice for on-site detection of prometryn in different kinds of herbs. Graphical abstract.

Development of a simple and rapid method for the detection of isomiroestrol in Pueraria candollei by an immunochromatographic strip test.

Pueraria candollei (P. candollei) is a traditional Thai herb widely used for estrogen replacement therapy because it contains many unique chromenes that possess potent estrogenic activity, one of which is known as isomiroestrol. Since isomiroestrol is a promising compound that is solely present in P. candollei, it can be used as an identifying marker for standardization of P. candollei. Here, we developed a lateral-flow immunochromatographic strip (ICS) test using a colloidal gold nanoparticle-conjugated anti-isomiroestrol monoclonal antibody (12C1-mAb) for the detection of isomiroestrol in plant samples and products of P. candollei. The advantages of the developed ICS over an enzyme-linked immunosorbent assay are its simplicity and rapidity, as the ICS test can be completed 15 min after dipping the strip into the analyte solution. The detectable concentration of isomiroestrol was 7.0 µg/mL. Considering the demand for the standardization of P. candollei due to concerns regarding its quality, our ICS test using isomiroestrol as an identifying marker would be effective and useful to assess the presence of isomiroestrol.

Development of a colloidal gold-based lateral flow dipstick immunoassay for rapid detection of chlorogenic acid and luteoloside in Flos Lonicerae Japonicae.

Flos Lonicerae Japonicae (FLJ), known as golden-and-silver honeysuckle, is a widely used Chinese herbal medicine with pharmacological activities and edibleness in China. Chlorogenic acid (CGA) and luteoloside are the quality control markers of FLJ regulated by the Chinese Pharmacopoeia (2015 edition). For rapid evaluation of the quality of FLJ, dipsticks for CGA and luteoloside detection were developed. The detection limit of the dipsticks for CGA and luteoloside, defined as the lowest concentration of the target analyte between which the test line was invisible, were 100 ng/mL and 200 ng/mL, respectively. The dipsticks were used for determination of CGA and luteoloside contents in FLJ, and the results were confirmed by high-performance liquid chromatography. The developed dipsticks, with their simplicity of use, lack of dependence on instruments and environmental friendliness, could be used to evaluate the quality of FLJ within 10 min.

Development of a colloidal gold nanoparticle-based immunochromatographic strip for the one-step detection of miroestrol and puerarin.

Pueraria candollei or White Kwao Krua (Leguminosae) is an indigenous plant in Thailand which has long been used in Thai traditional medicine. The tuberous root of this plant is widely used for rejuvenation, particularly in elder women. Among the bioactive compounds in P. candollei, miroestrol and puerarin exhibit estrogenic activity. This study aims to develop an immunochromatographic strip (ICS) with a colloidal gold-based detection system for the simultaneous detection of miroestrol and puerarin in a one-step analysis. The developed method is sensitive and specific for the detection of miroestrol and puerarin in raw materials and marketed products. The detection limits of miroestrol and puerarin were 0.15 and 4.5 µg, respectively. In addition, the results from the developed ICS were confirmed with an enzyme-linked immunosorbent assay and presented a good correlation between these two methods. This is the first report on the development of an ICS that can detect miroestrol and puerarin in one step. The developed ICS provides a simplified method for the detection of miroestrol and puerarin in P. candollei and Pueraria spp.

Antimicrobial efficacy of drug blended biosynthesized colloidal gold nanoparticles from Justicia glauca against oral pathogens: A nanoantibiotic approach.

Antimicrobial resistance is a challenging task for researchers to develop new strategies. Green synthesis of AuNPs is an eco-friendly approach, which can be utilized in the microbistatic and microbicidal activities. The current study is focused on Justicia glauca (aqueous leaf extract) mediated AuNPs synthesis at room temperature by treating chloroaurate ions, that shows an antagonistic effect with Azithromycin (AZM) and Clarithromycin (CLR) antibiotics against oral pathogenic bacteria and fungi (Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans, Lactobacillus acidophilus, Escherichia coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Candida albicans). Characterization of green synthesized AuNPs was done by using Ultraviolet-visible (UV-vis) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD) analysis and Energy Dispersive X-ray analysis (EDAX). Biosynthesized AuNPs were stable, hexagonal and spherical shaped with a size ∼32.5 ± 0.25 nm. The AuNPs and drug conjugated AuNPs showed potential antibacterial and antifungal activity against the oral pathogens. Minimum Inhibitory Concentration (MIC) values of biogenic AuNPs were observed in the range of 6.25-25 µg/mL against selected oral pathogens. Overall, we conclude that biogenic drug delivery system for AZM and CLR can be exploited as potential antimicrobial therapy in future, subject to detailed in-vitro and in-vivo cytotoxicity.

Generation and Application of Monoclonal Antibody Against Lycopene.

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

Polyphenol stabilized colloidal gold nanoparticles from Abutilon indicum leaf extract induce apoptosis in HT-29 colon cancer cells.

Green synthesized gold nanoparticles have received substantial attention owing to their biomedical applications, particularly in cancer therapy. Although anticancer activities of green synthesized gold nanoparticles have been reported earlier, the underlying mechanism behind their anticancer activity is still to be understood. The present study, describes the green synthesis of Abutilon indicum gold nanoparticles (AIGNPs) from Abutilon indicum leaf extract (AILE) and their cytotoxic mechanism in colon cancer cells. Dimensions of spherical shaped AIGNPs were found to be in the range of 1-20nm as determined by TEM. GC-MS and FTIR analysis indicated the presence of polyphenolic groups in AILE, which might have been involved in the stabilization of AIGNPs. In vitro free radical scavenging analysis revealed the radical quenching activity of AIGNPs. Further, the AIGNPs exhibited cytotoxicity in HT-29 colon cancer cells with IC50 values of 210 and 180µg/mL after 24 and 48h. This was mediated through nuclear morphological changes and cell membrane damage as evidenced by acridine orange/ethidium bromide, propidium iodide and AnnexinV-Cy3 staining methods. Mechanism of the observed cytotoxicity of AIGNPs was explained on the basis of increased levels of reactive oxygen species and simultaneous reduction in cellular antioxidants, which might have caused mitochondrial membrane potential loss, DNA damage and G1/S phase cell cycle arrest. Expression of cleaved Caspase-9, Caspase-8, Caspase-3, Lamin A/C and PARP, provided the clues for the induction of intrinsic and extrinsic apoptosis pathways in AIGNPs treated HT-29 cells. The study provides a preliminary guidance towards the development of colon cancer therapy using green synthesized gold nanoparticles.

[Advancement of colloidal gold chromatographic technique in screening of ochratoxin A].

Ochratoxin A (OTA) is a toxic secondary metabolite mainly produced by Aspergillus and Penicillium species, existing in a variety of foodstuffs and Chinese medicines. OTA is difficult to be detected in practice because of the characteristics such as trace amounts, toxicity, existing in complex matrices. In the numerous detection technologies, colloidal gold chromatographic techniques are highly sensitive, specific, cost-effective and user-friendly, and are being used increasingly for OTA screening. Recently, with the development of aptamer technology and its application in chromatographic technique, a newly colloidal gold aptamer chromatographic technique has been developed. This review elaborates the structures and principles of both traditional and newly colloidal gold chromatographic techniques, focuses on newly colloidal gold aptamer chromatographic technique, summarizes and compares their use in rapid detection of OTA. Finally, in order to provide a reference for better research of related work, the development trends of this novel technique are prospected.

Biosynthesis of gold nanoparticles using catclaw buttercup (Radix Ranunculi Ternati) and evaluation of its colloidal stability.

The biosynthesis of gold nanoparticles using catclaw buttercup (Radix Ranunculi Ternati) and their stability have been reported in this paper. The aqueous catclaw buttercup was used as mild reducing agent for gold nanoparticles synthesis from HAuCl4 solutions. The influence of reaction time, temperature and mass ratio of HAuCl4/catclaw buttercup were evaluated to investigate their effects on gold nanoparticles synthesis. Under the optimized reaction parameters, the gold nanoparticles obtained are characterized by UV-vis spectrum, X-ray diffraction (XRD), EDAX technique (EDX), high resolution transmission electron microscopy (HRTEM), FTIR spectrum, anthrone-sulfuric colorimetric method, and plus Improved-Lowry Protein Assay Kit. The HRTEM images showed that the biosynthesized gold nanoparticles are mostly spherical with size range from 9-24 nm. Furthermore, it was found that the biosynthesized gold nanoparticles possessed outstanding colloid stability in aqueous solutions as a function of category and concentration of monovalent salt and pH value of the solution when compared with chemosynthetic ones with the similar size. Anthrone-sulfuric colorimetric method revealed that there is no sugar in the biosynthesized gold colloid. While Improved-Lowry tests results demonstrated that the existence of much protein in the biosynthesized gold colloid, which may played an important role in stabilization of it. Owing to their stability, biocompatibility, lower cost and so on, gold nanoparticles synthesized by this biosynthesis method show potential application prospect in optoelectronic and biomedicine.

Nanoparticle preconditioning for enhanced thermal therapies in cancer.

Nanoparticles show tremendous promise in the safe and effective delivery of molecular adjuvants to enhance local cancer therapy. One important form of local cancer treatment that suffers from local recurrence and distant metastases is thermal therapy. In this article, we review a new concept involving the use of nanoparticle-delivered adjuvants to 'precondition' or alter the vascular and immunological biology of the tumor to enhance its susceptibility to thermal therapy. To this end, a number of opportunities to combine nanoparticles with vascular and immunologically active agents are reviewed. One specific example of preconditioning involves a gold nanoparticle tagged with a vascular targeting agent (i.e., TNF-α). This nanoparticle embodiment demonstrates preconditioning through a dramatic reduction in tumor blood flow and induction of vascular damage, which recruits a strong and sustained inflammatory infiltrate in the tumor. The ability of this nanoparticle preconditioning to enhance subsequent heat or cold thermal therapy in a variety of tumor models is reviewed. Finally, the potential for future clinical imaging to judge the extent of preconditioning and thus the optimal timing and extent of combinatorial thermal therapy is discussed.

Immunochromatographic assay for the detection of pseudojujubogenin glycosides.

INTRODUCTION: Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts. OBJECTIVE: The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody. METHODOLOGY: The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane. RESULTS: The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL. CONCLUSION: The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.

One-step purification of lectins from banana pulp using sugar-immobilized gold nano-particles.

To obtain lectins without tedious purification steps, we developed a convenient method for a one-step purification of lectins using sugar-immobilized gold nano-particles (SGNPs). Proteins in crude extracts from plant materials were precipitated with 60% ammonium sulphate, and the precipitate was re-dissolved in a small volume of phosphate buffer. The resultant solution was then mixed with appropriate SGNPs under an optimized condition. After incubating overnight at 4 degrees C, lectins in the mixture formed aggregate with SGNPs, which was visually detected and easily sedimented by centrifugation. The aggregate was dissolved by adding inhibitory sugars, which were identical to the non-reducing sugar moieties on the SGNPs. According to SDS-PAGE and MS of thus obtained proteins, it was found that SGNPs isolated lectins with a high purity. For example, a protein isolated from banana using Glcalpha-GNP (alpha-glucose-immobilized gold nano-particle) was identified as banana lectin by trypsin-digested peptide-MS finger printing method.

Development of a one-step immunochromatographic strip test for the detection of sennosides A and B.

An immunochromatographic strip test was developed to detect sennoside A (1) and sennoside B (2) using anti-1 and anti-2 monoclonal antibodies. The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of colloidal gold particles coated with the respective sennoside antibodies. The capture reagents were 1- and 2-human serum albumin (HSA) conjugates immobilised on a nitrocellulose membrane on the test strip. The sample containing 1 and 2, together with detector reagent, passed over the zone where the capture reagents had been immobilised. The analytes in the sample competed for binding to the limited amount of antibodies in the detector reagent with the immobilised 1- and 2-HSA conjugates on the membrane and hence positive samples showed no colour in the capture spot zone. Detection limits for the strip test were 125 ng/mL for both sennosides. The assay system is useful as a rapid and simple screening method for the detection of 1 and 2 in plants, drugs and body fluids.

Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications.

Submicroscopic gold particle suspensions scatter colored light when illuminated with white light, and we have observed that a light-scattering gold particle suspension has the same appearance as a fluorescing solution. Thus, when illuminated by a narrow beam of white light, a 40-nm gold sol displays a clear (not cloudy), green scattered light (Tyndall) beam and has the same appearance as a fluorescing fluorescein solution. These, as well as other, observations have suggested to us that, in general, light-scattering particles can be treated as fluorescent analogs and used as fluorescent analog tracers in immuno- and DNA probe assays as well as in cell and molecular biology studies. Light-scattering particles are advantageous in these applications because particles such as gold and silver have very high light-scattering powers, which allows these particles to be easily detected, by light-scattering, at particle concentrations as low as 10(-16) M. The scattered light can be detected by the unaided eye for qualitative measurements or with a simple light-sensitive detector for quantitative measurements. Moreover, individual particles can be easily detected by eye or a video camera using a simple light microscope with a proper illuminating system. In addition, submicroscopic particles which scatter blue, green, yellow, orange, or red light can be readily synthesized. Antibodies, DNA probes, and other tracer substances can be readily attached to gold and other particles without altering their light-scattering properties. In this article we present the theory which allows one to predict the light-scattering properties of particles of different sizes and compositions and identify those particle sizes and compositions which appear most adequate for particular applications. Furthermore, we calculate molar extinction coefficients and emission efficiencies for particles of different sizes and compositions which allows us to compare the light-producing powers of these particles with those of well known fluorescent tracers. A 60-nm gold particle, for example, is equivalent to about 3 x 10(5) fluorescein molecules. Very simple, easy to use, low-cost, ultrasensitive immuno- and DNA probe assays can be developed using light-scattering particles as fluorescent analog tracers. Single particles can be detected on cell surfaces and inside cells using light microscopy techniques with proper illumination as described in the article. At high particle densities, particle-labeled cells have the same appearance as fluorescent cells.

Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications.

In a companion article we present the idea that submicroscopic light-scattering particles, such as gold and silver particles, can be used as fluorescent analog tracers in biological and clinical applications. The light-emitting power and scattered light color of the particles can be adjusted by changing particle composition or diameter. Using Rayleigh and Mie light-scattering theory, we calculated the absorbance and light-scattering properties and power of particles of different diameters and selected compositions and compared them to the properties of fluorophores. In the present article, we evaluate experimentally the optical properties of particles of selected compositions and sizes and compare the results with the theoretically calculated values. We also discuss the methods which we use to measure the light-scattering properties of particles in suspension and to view individual particles by light microscopy. We also outline examples which demonstrate the use of light-scattering particles as fluorescent analogs in biological and clinical applications.