Optical biosensor for pharmaceuticals, antibiotics, hormones, endocrine disrupting chemicals and pesticides in water: Assay optimization process for estrone as example

Certain contaminants at trace concentrations in surface waters can have dramatic effects on the hormonal system of organisms in the aquatic environment. Therefore, immunoanalytical methods at a very low limit of detection (LOD) and a low limit of quantification (LOQ) are becoming more and more important for environmental analysis and especially for monitoring drinking water quality. Environmental monitoring of antibiotics, hormones, endocrine disrupting chemicals, and pesticides in real water samples (e.g. surface, ground or drinking water) with difficult matrices places high demands on chemical analysis. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe an assay optimization process with a fully automated immunoassay for estrone which resulted in a LOD below 0.20 ng L⁻¹ and a LOQ below 1.40 ng L⁻¹. In contrast to common analytical methods such as GC-MS or HPLC-MS, the biosensor used requires no sample pre-treatment and pre-concentration. The very low validation parameters for estrone are the result of the continuous optimization of the immunoassay. The basis of our sensitive assay is the antibody with a high affinity constant towards estrone. During the optimization process, we reduced the amount of antibody per sample and improved the chip surface modification. Finally, this proceeding led to a calibration routine with an amount of antibody of only 3.0 ng per sample (sample volume: 1.0 mL). The reduction of the amount of antibody per sample results in better validation parameters (LOD, LOQ, and IC₅₀), but this reduction leads to the current device-related limitation of the River Analyser (RIANA).For some endocrine disrupting compounds, no effect levels (NOELs) in the lower nanogram per liter range are reported. This defines the challenge, which analytical methods have to compete with and our RIANA instrument with its improved sensitivity for the detection of a single hormone in the lower nanogram per liter range is a powerful tool in aquatic analytics in addition to the common analytical methods.     

Fluorimetric assay for measuring Dns-His-Lys-Arg-His-Lys cleaving enzyme using high-performance liquid chromatography

A rapid and sensitive assay for the determination of Dns-His-Lys-Arg-His-Lys cleaving enzyme activity is reported. This assay is based on fluorimetric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys-Arg-His-d-Lys, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This assay is sensitive enough to measure 5-dimethylaminonaphthalene-1-sulfonyl-His-Lys at concentrations as low as 7 pmol, and yields highly reproducible results and requires less than 9.0 min per sample for separation and quantitation. The optimum pH for Dns-His-Lys-Arg-His-Lys cleaving enzyme activity was 7.5-8.0. The Michaelis constant (K_m) and the maximum velocity (V_max) values were 33.3 μM and 47.07 pmol/(μg h), respectively with the use of enzyme extract obtained from bovine pituitary. By using this assay, axonal transport of this enzyme activity was observed 48 h after double ligations of rat sciatic nerves. The high sensitivity and selectivity of this assay would be useful for clarification of the physiological role of this enzyme.     

Development of an automated sequential injection on-line solvent extraction-back extraction procedure as demonstrated for the determination of cadmium with detection by electrothermal atomic absorption spectrometry

An automated sequential injection (SI) on-line solvent extraction-back extraction separation/preconcentration procedure is described. Demonstrated for the assay of cadmium by electrothermal atomic absorption spectrometry (ETAAS), the analyte is initially complexed with ammonium pyrrolidinedithiocarbamate (APDC) in citrate buffer and the chelate is extracted into isobutyl methyl ketone (IBMK), which is separated from the aqueous phase by means of a newly designed dual-conical gravitational phase separator. A metered amount of the organic eluate is aspirated and stored in the PTFE holding coil (HC) of the SI-system. Afterwards, it is dispensed and mixed with an aqueous back extractant of dilute nitric acid containing Hg(II) ions as stripping agent, thereby facilitating a rapid metal-exchange reaction with the APDC ligand and transfer of the Cd into the aqueous phase. The aqueous phase is separated in a second dual-conical gravitational phase separator, and 30 μl of it is entrapped and metered in a sample loop (SL) and subsequently introduced via air segmentation into the graphite tube for analyte quantification. The ETAAS determination is performed in parallel with the separation/preconcentration process of the ensuing sample. An enrichment factor of 21.4, a detection limit of 2.7 ng l⁻¹, along with a sampling frequency of 13 h⁻¹ were obtained at a sample flow rate of 6.0 ml min⁻¹. The precision (R.S.D.) at the 0.4 μg l⁻¹ level was 1.8% as compared to 3.2% when quantifying the organic extractant directly. The applicability of the procedure is demonstrated for the determination of trace levels of cadmium in three certified reference materials.     

Selective flow injection analysis of ultra-trace amounts of Cr(VI), preconcentration of it by solvent extraction, and determination by electrothermal atomic absorption spectrometry (ETAAS)

A rapid, robust, sensitive and selective time-based flow injection (FI) on-line solvent extraction system interfaced with electrothermal atomic absorption spectrometry (ETAAS) is described for analyzing ultra-trace amounts of Cr(VI). The sample is initially mixed on-line with isobutyl methyl ketone (IBMK). The Cr(VI) is complexed by reaction with ammonium pyrrolidine dithiocarbamate (APDC), and the non-charged Cr(VI)–PDC chelate formed is extracted into IBMK in a knotted reactor made from PTFE tubing. The organic extractant is separated from the aqueous phase by a gravity phase separator with a small conical cavity and delivered into a collector tube, from which 55 μl organic concentrate is subsequently introduced via an air flow into the graphite tube of the ETAAS instrument. The operations of the FI-system and the ETAAS detector are synchronously coupled. A significant advantage of the approach is that matrix constituents, such as high salt contents, effectively are eliminated. The extraction procedure was optimized by a simplex approach. A central composite design was subsequently employed to verify the estimated operational optimum. An 18-fold enhancement in sensitivity of Cr(VI) was achieved after preconcentration for 99 s at a sample flow rate of 5.5 ml min⁻¹, as compared to direct introduction of 55 μl of sample, yielding a detection limit (3σ) of 3.3 ng l⁻¹. The sampling frequency was 24.2 samples h⁻¹. The proposed method was successfully evaluated by analyzing a NIST Cr(VI)-reference material, synthetic seawater and waste waters, and waste water samples from an incineration plant and a desulphurization plant, respectively.     

Development and Use of Antibodies in Surface Plasmon Resonance-Based Immunosensors for Environmental Monitoring

The interaction between antibody and antigen is characterised by relatively high affinity and specificity, making this type of reaction a prime candidate for use as an analytical tool. The interaction may be combined with biosensors in the production of immunosensors for environmental monitoring. Polyclonal and monoclonal antibodies have had a significant impact in analytical detection systems over the past few decades with antibody fragments becoming important in recent years. Production of antibodies to small haptens requires the initial conjugation of hapten to a larger carrier molecule. Once hapten-carrier conjugates have been produced, polyclonal, monoclonal and various antibody fragments may be produced by differing protocols. A critical step in the production of antibody fragments is the development of efficient screening procedures to identify suitable antibody-producing clones and this has been reviewed in this article. Various antibody types may then be used in the generation of immunosensors for the monitoring of environmental pollutants. The selection of the appropriate sensor technology applicable for the determination of an antibody-antigen interaction is of prime importance for immunosensor development. One example of such an application is surface plasmon resonance-based biosensors, as they provide real-time analysis of interactions between the antibody and antigen of interest.     

Genotoxicity and Cytotoxicity of 4-Nonylphenol Ethoxylate on Lymphocytes as Assessed by the Comet Assay

Surfactants, which are prevalent at industrial sites and in the environment generally, are potential risk factors in human carcinogenesis. The widespread industrial use of surfactants such as 4-alkylphenol ethoxylates and their prevalence in many cleaning products have provoked studies about surfactant concentrations in water and their toxicity levels. Up to now, these substances have mainly been tested on aquatic organisms. Though tests on human cell lines are rare. The alkaline Comet assay was performed to evaluate the genotoxicity of 4-nonylphenol ethoxylate, a biodegradable product of 4-alkylphenol ethoxylate, in human lymphocytes. Concentrations tested ranged from 0.15 to 150 µg/mL. Test concentrations of 10 to 15 µg/mL caused an increase level of DNA migration in human cells, but without inducing excessive toxicity (viability > 80%). Though induced levels of DNA migration starting at concentrations of 30 µg/mL may have been due to excessive levels of cytotoxicity (viability < 70%). Based on these data, 4-nonylphenol ethoxylate can induce DNA damage in human lymphocytes but at higher concentrations than are normally found in river or drinking water. However, considering the prevalence of surfactants, the measured genotoxicity of these substances is of concern. Further investigations on human target cells are necessary to evaluate the carcinogenic impact of surfactants and reconsider their environmental acceptance.     

Development of a competitive ELISA for the quantification of F5 conjugate in HER2-targeted STEALTH immunoliposome doxorubicin in plasma samples

HER2 (human epidermal growth factor receptor 2, erbB2, or neu) is overexpressed by a large number of tumor types and has been identified as an important target for cancer therapy. F5 is a single-chain human antibody fragment that recognizes HER2 receptor and is covalently conjugated to PEGylated lipid to form F5 conjugate (F5CG) in the product HER2 targeted STEALTH immunoliposome doxorubicin. Here we described the method development of a competitive enzyme-linked immunosorbent assay (ELISA) for the determination of total concentration of F5 conjugate in plasma samples. The method involved the biotinylation of F5CG, detergent treatment of plasma sample to solubilize F5CG into monomeric form, and competitive ELISA for solubilized F5CG competitively binding to anti-F5CG antibody with biotinylated F5CG for the determination of total F5CG in plasma. The detection range of this method was from 0.2 ng/mL to 125 ng/mL for F5CG in plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL. This method was established and used for the measurement of F5CG concentration to provide information about F5CG circulation after the administration of immunoliposome in preclinical studies.      

内蒙古部分地区猪圆环病毒2型(PCV-2)感染的血清学调查

应用酶联免疫吸附实验(ELISA)方法对内蒙古部分地区猪场的182份猪血清进行猪圆环病毒2型(PCV-2)感染的抗体检测,其中包括母猪、种公猪、哺乳仔猪、生长育肥猪、保育猪.平均阳性感染率为39.6%(72/182),母猪阳性感染率为44.9%(22/49),种公猪阳性感染率为73.7%(14/19),生长育肥猪阳性感染率为38.8%(19/49),保育猪阳性感染率为40.5%(17/42),哺乳仔猪全部为阴性(15).实验检测结果证实,内蒙古地区养猪场存在猪圆环病毒II型(PCV-2)感染,而且比较严重.