Candace R. S. Bever

Impedance Biosensors: Applications to Sustainability and Remaining Technical Challenges

Due to their all-electrical nature, impedance biosensors have significant potential for use as simple and portable sensors for environmental studies and environmental monitoring. Detection of two endocrine-disrupting chemicals (EDC), norfluoxetine and BDE-47, is reported here by impedance biosensing, with a detection limit of 8.5 and 1.3 ng/mL for norfluoxetine and BDE-47, respectively. Although impedance biosensors have been widely studied in the academic literature, commercial applications have been hindered by several technical limitations, including possible limitations to small analytes, the complexity of impedance detection, susceptibility to nonspecific adsorption, and stability of biomolecule immobilization. Recent research into methods to overcome these obstacles is briefly reviewed. New results demonstrating antibody regeneration atop degenerate (highly doped) Si are also reported. Using 0.2 M KSCN and 10 mM HF for antibody regeneration, peanut protein Ara h 1 is detected daily during a 30 day trial.

Heterologous antigen selection of camelid heavy chain single domain antibodies against tetrabromobisphenol A.

Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules.

Development and Utilization of Camelid VHH Antibodies from Alpaca for 2,2′,4,4′-Tetrabrominated Diphenyl Ether Detection

An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies.

One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein

Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environmental and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. The ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL(-1). Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogues was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP-based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples via this one-step assay ranged from 96.7% to 109.9% and correlated well with a high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS) method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.

Method for Sorting and Pairwise Selection of Nanobodies for the Development of Highly Sensitive Sandwich Immunoassays

Single domain heavychain binders (nanobodies) obtained from camelid antibody libraries hold a great promise for immunoassay development. However, there is no simple method to select the most valuable nanobodies from the crowd of positive clones obtained after the initial screening. In this paper, we describe a novel nanobody-based platform that allows comparison of the reactivity of hundreds of clones with the labeled antigen, and identifies the best nanobody pairs for two-site immunoassay development. The output clones are biotinylated in vivo in 96-well culture blocks and then used to saturate the biotin binding capacity of avidin coated wells. This standardizes the amount of captured antibody allowing their sorting by ranking their reactivity with the labeled antigen. Using human soluble epoxide hydrolase (sEH) as a model antigen, we were able to classify 96 clones in four families and confirm this classification by sequencing. This provided a criterion to select a restricted panel of five capturing antibodies and to test each of them against the rest of the 96 clones. The method constitutes a powerful tool for epitope binning, and in our case allowed development of a sandwich ELISA for sEH with a detection limit of 63 pg/mL and four log dynamic range, which performed with excellent recovery in different tissue extracts. This strategy provides a systematic way to test nanobody pairwise combinations and would have a broad utility for the development of highly sensitive sandwich immunoassays.

Effects of triclosan in breast milk on the infant fecal microbiome

Triclosan is frequently used for its antimicrobial properties and has been detected in human serum, urine, and breast milk. Animal and molecular studies have shown that triclosan exerts a wide range of adverse health effects at both high (ppm) and low (ppb) concentrations. Since triclosan is of growing concern to human and environmental health, there is a need to improve extraction procedures and to study additional effects from triclosan exposure. In this study, we have improved triclosan extraction from breast milk by using salt (MgSO4) to reduce emulsion formation and increase water polarity and water (∼80%) to enhance the overall extraction efficiency (∼3.5 fold). This extraction method was applied to breast milk samples collected from donors who i) recorded their use of triclosan-containing personal care products and ii) provided matching infant stool samples. Of the participants who had detectable amounts of triclosan in their breast milk, nine (75%) of them reported daily use of triclosan-containing personal care products. Levels of triclosan in breast milk were compared to the donors infants fecal microbiome. We found that the bacterial diversity in the fecal microbiome of the infants exposed to breast milk with detectable triclosan levels differed compared to their peers exposed to milk containing non-detectable amounts. This finding implies that exogenous chemicals are impacting microbiome diversity.

A New Conjugation Method Used for the Development of an Immunoassay for the Detection of Amanitin, a Deadly Mushroom Toxin

One of the deadliest mushrooms is the death cap mushroom, Amanita phalloides. The most toxic constituent is α-amanitin, a bicyclic octapeptide, which damages the liver and kidneys. To develop a new tool for detecting this toxin, polyclonal antibodies were generated and characterized. Both α- and β-amanitin were coupled to carrier proteins through four different linking chemistries, one of which has never before been described. These conjugates were evaluated for their effectiveness in generating antibodies specific for the free toxin, as well as their utility in formatting heterogeneous assays with high sensitivity. Ultimately, these efforts yielded a newly described conjugation procedure utilizing periodate oxidation followed by reductive amination that successfully resulted in generating sensitive immunoassays (limit of detection (LOD), ~1.0 µg/L). The assays were characterized for their selectivity and were found to equally detect α-, β-, and γ-amanitin, and not cross-react with other toxins tested. Toxin detection in mushrooms was possible using a simple sample preparation method. This enzyme-linked immunosorbent assay (ELISA) is a simple and fast test, and readily detects amatoxins extracted from A. phalloides.