Abstract
In this study, two mutant strains, TBC and TBC+, able to biosynthesize novel functional magnetosome-nanobody (Nb) were derived from the magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1. The magnetosome-Nbs biosynthesized by TBC+ containing multi-copies of Nb gene had higher binding ability to an environmental pollutant tetrabromobisphenol A (TBBPA) than those biosynthesized by TBC containing only one copy of Nb gene. The magnetosome-Nbs from TBC+ can effectively bind to TBBPA in solutions with high capacity without being affected by a broad range of NaCl and methanol concentrations as well as pH. Therefore, a magnetosome-Nb-based enzyme-linked immunosorbent assay (ELISA) was developed and optimized for the detection of TBBPA, yielding a half-maximum signal inhibition concentration of 0.23 ng/mL and a limit of detection of 0.025 ng/mL. The assay was used to detect TBBPA in spiked river water samples, giving average recoveries between 90 and 120% and coefficients of variation 2.5–6.3%. The magnetosome-Nb complex could be reused 4 times in ELISA without affecting the performance of the assay. Our results demonstrate the potential of magnetosome-Nbs produced by TCB+ as cost-effective and environmental-friendly reagents for immunoassays to detect small molecules in environmental waters.
Keywords: Environmental monitoring, Enzyme-linked immunosorbent assay, Magnetosome, Magnetospirillum gryphiswaldense, Nanobody, Tetrabromobisphenol A
Introduction
Magnetosomes are magnetic nanoparticles biosynthesized by a group of magnetotactic bacteria (MTB). Compared with other MTBs, the alpha-proteobacterium Magnetospirillum gryphiswaldense MSR-1 can offer the largest amount of magnetosomes of those surveyed. A complex genetically controlled process leads to the formation of colloidally stable particles naturally covered by a phospholipid bilayer containing specific proteins, with high magnetism, a narrow size distribution, and uniform morphology. Recently, a study revealed that magnetosomes are apparently safe and do not cause any potential risk to the environment compared to physio-chemical abiotic synthetic magnetic nanoparticles [1]. These properties suggest that magnetosomes could be ideal reagents for biotechnological applications, e.g., magnetosome-based immunoassays [2–4].
However, the applications of magnetosomes in biotechnology usually require incorporation of functionalities to the particles, such as chemical modifications. Chemical coupling often lacks selectivity and requires harsh conditions. On the other end, bioactive proteins can be expressed on magnetosomes as a genetic fusion with an integral magnetosome membrane protein under physiological conditions in the presence of a variety of chaperons. The highly abundant membrane protein mamC with non-essential and redundant functions often serves as anchors for fluorophores, antibodies, and a variety of receptors and enzymes [5–9]. X-ray crystallography showed that mamC contains two integral transmembrane helices with an acidic intraluminal helical loop, which has two charge-separated regions binding to magnetite particles [10]. However, fusing and expressing a large protein like an antibody to mamC is complicated and difficult. On the other end, the heavy chain variable domains of heavy-chain antibodies (VHHs), also known as nanobodies (Nbs), arise from a short encoding gene (~360 bp), so they are easy to express in a prokaryotic and eukaryotic expression system requiring no post-translational modifications [11]. Thus, Nbs can be easily complexed with mamC. Such approach was recently used for the biosynthesis in MSR-1 of magnetosome-Nb complexes specific for the insecticide fipronil employed to develop an ELISA for the detection of fipronil [12].
Most Nb applications have been for proteins and other large molecules. However, Nbs are attractively employed in the detection of small compounds from various matrices due to their inherent traits, such as high solubility, high avidity for the targeted hapten, and high resilience to solvent and temperature [13–15]. For example, Nbs against tetrabromobisphenol A (TBBPA) were recently obtained [16]. TBBPA is one of the most widely used brominated flame retardants for preventing or reducing the flammability and combustibility of polymers and textiles with a production volume of 170,000 tons as in 2004 [17,18]. Unlike bis-phenol A, the phenols of TBBPA are sterically hindered and it is poorly metabolized. TBBPA can easily enter into the environment via abrasion or volatilization and was widely detected in biotic and abiotic matrixes [19–21]. TBBPA can induce neurotoxicity, immunotoxicity, cytotoxicity, and disruption of endocrine function [22,23]. So, it is very meaningful to develop diverse analytical tools to monitor TBBPA for early prevention of environmental exposure.
To investigate the general applicability of magnetosome-Nbs in the field of bioanalytical technology, herein, Nbs against TBBPA were genetically fused with mamC of magnetosomes and used to develop an immunoassay for the analysis of this compound in water samples.
Materials and Methods
Bacterial strains and culture conditions
The bacterial strains and plasmids used in this study are listed in Table S1. E. coli strains were cultured in Luria broth (LB) at 37 °C. MSR-1 was cultured in serum bottles filled with 50 mL of sodium lactate medium (SLM) with 20 μM ferric citrate or sodium glutamate medium (replace NH4Cl and yeast extract in SLM with 4 g sodium glutamate) as described previously [24]. Large-scale MSR-1 cells (5.0 L) were fed-batch cultured in a 7.5-L fermenter according to the method previously reported [25]. Antibiotic concentrations in culture media were prepared as follows: for E. coli, ampicillin at 100 μg/mL and kanamycin at 50 μg/mL; for MSR-1, kanamycin at 5 μg/mL and nalidixic acid at 5 μg/mL. The growth of MSR-1 (OD565) and magnetic response (Cmag) in the fermenter were detected as the method described previously [26].
Construction of recombinant plasmids and strains
All primers used in this study are listed in Table S2. E. coli strains DH5α and S17–1 were respectively used for DNA cloning and conjugation transfer of target genes. The enzymes employed were purchased from Takara Co., Ltd. (Beijing, China). PCR reaction was performed using 2 × Phanta Max Master Mix (Vazyme, Nanjing, China). mamC and its upstream and downstream flanking fragments were amplified from MSR-1 genomic DNA. Anti-TBBPA Nb gene with 6×his tag at the C-terminus was amplified from plasmid (pecan 45) containing the VHH-alkaline phosphatase gene [27]. VHH and mamC were fused to generate a fusion gene, TBC, by PCR with primers TB-F (BamH I) and C-R (Xba I). Two plasmids were constructed as follows. (1) uTBCd, a cassette containing mamC’s upstream region, TBC, and mamC’s downstream region, was assembled by fusion PCR and then cloned into a plasmid vector pK18mobSacB to construct the suicide recombination plasmid pKTBC. Finally, pKTBC was transferred into E. coli S17–1 by heat shock and subsequently into the M. gryphiswaldense MSR-1 wild type (WT) strain by conjugation to construct a mutant strain TBC. (2) The fusion gene TBC was inserted into the polycloning site of a broad host range plasmid vector pBBR1MCS-2 (BamH I and Xba I) to create a recombinant plasmid pBBRTBC. This plasmid was transferred into the mutant strain TBC to obtain an engineering strain TBC+. All these recombinant strains and plasmids are listed in Table S1.
Extraction of magnetosome-Nb complexes
TBC+ cells (5.0 L) were fed-batch cultured in a fermenter as described previously [25] and harvested by centrifugation (Cmag = ~1.0). Magnetosome-Nb complexes were extracted as follows: a 1.0 g amount of cell pellets was suspended in 20 mL of 0.01 M phosphate buffered saline (PBS, pH 7.4) and disrupted ultrasonically for 30 min (work 3 s and stop 5 s every cycle) at 200 W (next to 100 W, 60 W in turn). The complexes were isolated from the cell homogenate with a magnet and resuspended in 20 mL of PBS, followed by ultrasonic exposure for 30 min at 60 W to reduce cytoplasmic proteins. The suspension was placed on a magnet to separate magnetosome-Nb complexes from the supernatant. The extraction procedure was repeated several times until the OD260 and OD280 of the supernatant were stable. The purity and size of magnetosome-Nbs were evaluated by transmission electron microscopy (TEM) (JEM-1400, Japan; CCD: Gatan 832, 4 k × 3.7 k, USA).
Magnetosome-Nb-based ELISA for TBBPA
Magnetosome-Nb-based ELISAs were carried out according to the method described previously with slight modifications [3]. Briefly, a 96-well microtiter plate was blocked with 1% gelatin (or skimmed milk power) dissolved in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C (300 μL per well). The microtiter plate was washed three times using PBST (PBS containing 0.05% Tween-20). Magnetosome-Nb complexes were blocked with 2% bovine serum albumin in PBS (pH 7.4) for 3 h at room temperature and then washed three times with PBST. For a competitive ELISA, an aliquot of magnetosome-Nbs was transferred to the blocked 96-well microtiter plate (100 μL per well). The solutions of TBBPA and hapten T5 (Fig. S1) conjugated to horseradish peroxidase (T5-HRP), each at 50 μL, were successively added to wells harboring magnetosome-Nbs and incubated for 1 h on an oscillator (150 rpm/min). The plate was then fastened on magnetic frame and washed three times using PBST. A 100-μL aliquot of substrate solution (400 μL of 0.6% 3,3’,5,5’-tetramethylbenzidine and 100 μL of 1% H2O2 in 25 mL of citrate buffer, pH 5.5) was added into the wells and the reaction was stopped at 10 min by adding 50 μL of H2SO4 (2 M). Finally, the absorbance was read at 450 nm on a microtiter plate reader (ELx800, Bio Tek, USA).
Analysis of TBBPA in water samples
The resulting magnetosome-Nb-based ELISA was employed to detect TBBPA in water samples collected from Xiao Qing He river and Jingmi Diversion Canal in Beijing, China. Blank water samples were spiked with TBBPA at final concentrations of 0.1, 0.2, and 0.5 ng/mL. These samples were passed through a 0.22-μm filter (Waters, MA, USA) and subjected to ELISAs directly. TBBPA levels in the spiked samples were also detected according to a liquid chromatography tandem mass spectrometry (LC-MS/MS) method described by Yang et al. [28]. Finally, this ELISA was applied to the analysis of TBBPA in 12 real-world water samples which were collected from the same river (6 samples) and canal (6 samples) aforementioned.
Regeneration of the magnetosome-Nb complexes
Magnetosome-Nbs were regenerated using acidic media according to the procedure described previously [4]. Briefly, following the performance of ELISAs, magnetosome-Nbs binding to TBBPA were washed with 0.01 M PBS (pH 7.4) thoroughly and then incubated in H2SO4 solutions (pH 3) with shaking at 37 °C for 1 min. After another wash with PBS, the regenerated magnetosome-Nbs were employed in a new ELISA for TBBPA. The regeneration process, using the same method as described above, was repeated 6 times and the half-maximum signal inhibition concentration (IC50) and A0 (signal in the absence of TBBPA) of each ELISA for TBBPA using regenerated magnetosome-Nbs were compared with those using fresh one.
Results and Discussion
Construction of recombinant strains to biosynthesize magnetosome-Nbs
Because of its high abundance in the membrane and little influence on the biomineralization of magnetosomes, mamC was attractively used as an anchor protein to fuse with other functional materials. In order to obtain magnetosome-Nbs possessing desirable affinity to TBBPA, two recombinant strains, TBC and TBC+, that could biosynthesize magnetosomes with varying abundance of Nbs were constructed. The procedure of strain construction is shown in Fig. 1A. pKTBC carrying the gene cassette uTBCd expressing the anti-TBBPA Nb at the C-terminal of mamC was transferred into WT MSR-1 to generate a mutant strain, named as TBC. However, TBC has only one copy of Nb gene in its genome, which might result low level expression of Nbs on magnetosomes. Thus, to improve Nb expression, a multi-copy broad host range vector pBBR1MCS-2 carrying a fusion gene TBC, named pBBRTBC, was transferred into the mutant strain TBC, generating TBC+ with multi-copy Nb gene. A band of fusion protein mamC-Nb appeared at the position as expected in a Western-blot for membrane proteins of magnetosomes biosynthesized by TBC+ (Fig. 1B).
Fig. 1.
Construction of recombinant MSR-1 strains capable of biomineralizing magnetosome-Nbs. A: The schematic diagram of recombinant strain construction. B: Fusion protein mamC-Nb displayed on magnetosomes detected by Western-blot; 1: WT; 2: TBC+. C: Magnetosome-Nb complexes employed in a non-competitive ELISA. “***”: significant difference (p< 0.01).
The C-terminus of Nbs was site-directly oriented toward the surface of magnetosomes, which might facilitate the binding of Nbs to TBBPA by reducing chances of steric hindrance on the N-terminal hapten-binding site. In a non-competitive ELISA, the binding activity of magnetosome-Nbs from TBC+ to T5-HRP was significantly different from that of magnetosome from WT (Fig. 1C). Magnetosome-Nbs biosynthesized by TBC+ showed stronger binding ability to TBBPA than those biosynthesized by TBC in competitive ELISAs (Fig. S2), probably due to more Nbs expressed on magnetosomes in TBC+ than in TBC. Thus, magnetosome-Nb complexes from TBC+ were employed in the remainder of the study.
Fermentation of TBC+ cells
It has been reported that a high concentration of oxygen in culture media was good for MSR-1 growth, whereas a low level of dissolved oxygen (dO2<1%) was suitable for the biomineralization of magnetosomes [25]. The feature of magnetosomes could be evaluated by monitoring magnetic response, i.e. Cmag value. During fermentation, the OD565 and Cmag values of TBC+ cells in a 7.5-L fermenter were recorded.
The curve of magnetic response (Cmag) consisted of down, up and stable stages (Fig. 2). TBC+ cells were initially propagated in 500 mL of SLM containing 20 μM of ferric citrate, leading to the biosynthesis of magnetosome-Nbs. After being transferred into a fermenter (10% inoculation), a high concentration of dO2 inhibited the biomineralization of magnetosomes, leading to the drop of Cmag values in the first stage. About 26 h later, cells started to proliferate quickly and move into an exponential growth phase. Meanwhile, Cmag values increased rapidly, indicating the fast biosynthesis of magnetosomes. Finally, Cmag values came to a stable stage at ~32 h and kept above 1.0 for more than 70 h. In order to obtain high quality and quantity of magnetosome-Nbs, cells were harvested at 74 h although cell growth was still in the exponential phase (OD565 = 9.70). The yield of cells was about 83.6 g and the amount of magnetosome-Nbs from cells were 267.9 mg/g, all in wet weight (ww). TEM images revealed that most of the magnetosome-Nbs have a narrow size distribution of 30–50 nm in diameter (Fig. S3).
Fig. 2.
The growth (OD565) and magnetic response (Cmag values) curves of TBC+ with fed-batch culture in a fermenter.
Magnetosome-Nb-based ELISA for TBBPA
Magnetosomes were reported to possess intrinsic peroxidase-like activity, of which potential interferences with the tracer hapten-HRP employed in ELISAs can be minimized by using a limited amount of magnetosomes [29]. In this study, 200 μg/mL magnetosome-Nb/PBS suspension (100 μL per well) and 20 ng/mL T5-HRP (50 μL per well) were selected according to a checkerboard method for the next assay optimization.
Although TBBPA consists of two polar phenol groups, it is a lipophilic compound which has better solubility in organic solvents (e.g., methanol, ethanol and acetonitrile) than in aqueous solution. Methanol is commonly used as a solubilizer in immunoassays for lipophilic compounds. The performance of ELISA can be influenced by ionic strength, organic solvent, and pH of the assay buffer. To optimize a magnetosome-Nb-based ELISA for TBBPA, different calibration curves were generated in PBS with variables (NaCl, methanol, or pH). As shown in Fig. 3, magnetosome-Nbs exhibited effective binding abilities to TBBPA in PBS containing a broad range of NaCl and methanol concentrations as well as pH. ELISAs showed the highest sensitivities with 0.137 M NaCl, 5% methanol, and pH 9 among all the variables tested (Fig. 3). Therefore, a calibration curve was generated in PBS under the conditions optimized as above (Fig. 4A), with an IC50 of 0.23 ng/mL, a limit of detection (LOD, IC10) of 0.025 ng/mL, and a linear range (IC20-IC80) of 0.06–0.6 ng/mL. The magnetosome-Nb-based ELISA procedure is illustrated in Fig. 4B. The sensitivity of the resultant ELISA was 4.5- and 1.6-fold higher than the assay using magnetosome-Nbs constructed chemically via n-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (IC50=1.04 ng/mL) [3] and that using magnetosome-Nbs constructed via biotin and streptavidin (IC50=0.37 ng/mL) [4], respectively. In fact, both the SPDP method and the biotin–avidin method (glutaraldehyde was employed) involved the use of chemical cross-linkers, which probably affected the activity of membrane proteins and Nbs to some extent. In this study, genetic engineering techniques were employed to fuse Nbs with mamC protein, which might circumvent the influence of covalent coupling on Nb activity.
Fig. 3.
Optimization of magnetosome-Nb-based ELISA for TBBPA. The data are average of three replicates. A: TBBPA dissolved in PBS (pH 7.4) with NaCl at different concentrations. B: TBBPA dissolved in PBS (pH 7.4 and 0.137 M NaCl) containing different amounts of methanol. C: TBBPA dissolved in PBS (0.137 M NaCl and 5% methanol) with different pH.
Fig. 4.
The optimized magnetosome-Nb-based ELISA for TBBPA. A: Calibration curve of the magnetosome-Nb-based ELISA for TBBPA. TBBPA dissolved in PBS with 0.137 M NaCl, 5% methanol, and pH 9.0. The data are average of three replicates. B: Schematic diagram of the magnetosome-Nb-based ELISA for TBBPA.
Analysis of TBBPA in water by ELISA
The magnetosome-Nb-based ELISA was applied to detect TBBPA spiked in water samples at different levels. No matrix effect of water samples on the assay was observed and they were subjected to the ELISA directly. The average recoveries of TBBPA from the fortified water samples were in a range of 90–120%, with coefficients of variation (CVs) of 2.5–6.3% (Table 1). The results by these magnetosome-Nb-based ELISAs were correlated well to those by LC-MS/MS. The levels of TBBPA in all real-world water samples determined by the ELISAs were below the LOD (data not shown). Therefore, the developed immunomagnetic assay was demonstrated to be a valid method to detect TBBPA in waters.
Table 1.
Comparison of recoveries of TBBPA from water samples as determined by magnetosome-Nb-based ELISA and LC-MS/MS.
| Samples | Spiked level (ng/mL) | Detected by ELISA | Detected by LC-MS/MS | ||
|---|---|---|---|---|---|
|
| |||||
| Concentration (ng/mL) (± SD, n=3) | Average recovery (CV), % | Concentration (ng/mL) | Average recovery, % | ||
|
| |||||
| Xiao Jia He River | 0 | < LOD | < LOD | ||
| 0.1 | 0.11±0.007 | 110 (6.3) | 0.11 | 110 | |
| 0.2 | 0.23±0.007 | 115 (3.0) | 0.19 | 95 | |
| 0.5 | 0.45±0.018 | 90 (4.0) | 0.43 | 86 | |
| Jingmi Diversion Canal | 0 | < LOD | < LOD | ||
| 0.1 | 0.12±0.003 | 120 (2.5) | 0.11 | 110 | |
| 0.2 | 0.21±0.006 | 105 (2.9) | 0.18 | 90 | |
| 0.5 | 0.51±0.020 | 102 (3.9) | 0.49 | 98 | |
“LOD” indicates that the concentration of TBBPA in blank water samples was below the Limit of Detection using the detection method. “CV” stands for Coefficient of Variation (%).
LC-MS/MS is extensively employed to detect TBBPA in environmental samples. Compared to chromatographic methods, immunoassays are simple, cost-effective, high throughput, and suitable for on-site detection. Nb-based immunoassays were proven to be sensitive and selective for monitoring environmental compounds [30]. In some cases, the sensitivities of Nb-based ELISAs were even higher than those from polyclonal or monoclonal antibody-based immunoassays [31,32].
So far, Nbs have been immobilized on magnetosomes through multiple ways. Anti-TBBPA Nbs were chemically conjugated to magnetosomes with the bifunctional reagent SPDP [3] or biologically immobilized on magnetosomes through biotin and streptavidin [4]. In addition, Nbs can be directly displayed on magnetosomes by gene fusion using mamC as an anchor, as shown in the present and previous studies [12]. The resultant magnetosome-Nb complexes facilitated the ELISA performance obviously, illustrating the nanoparticles a promise in immunoassays for the detection of environmental compounds in real samples.
Reusability of magnetosome-Nbs
The binding of Nbs to TBBPA can be dissociated under harsh conditions (e.g., extreme pH) and restored by neutralization shortly as shown in our previous study [4]. In the present study, the dissociation of TBBPA from magnetosome-Nbs could be achieved by incubating the complexes of magnetosome-Nb-TBBPA in the acidic solution (pH 3) for 1 min. After neutralization, the magnetosome-Nbs were used for the detection of TBBPA and no statistic differences were observed between the performance of ELISAs (A0 and IC50) using the fresh immunomagnetic particles and that using the 1st–4th regenerated ones (Table 2). It is noted that the IC50 values increased as the particles from the 5th and 6th regeneration were used for ELISAs, indicating the binding activity of Nb was reduced after multiple acidic washes. The results suggested that robust magnetosome-Nbs could be reused at least 4 times.
Table 2.
Reusability of magnetosome-Nbs.
| Times of reuse | A0 | IC50 (ng/mL) |
|---|---|---|
|
| ||
| 0 (fresh material) | 1.188±0.05 a | 0.23±0.01 a |
| 1st | 1.207±0.08 a | 0.22±0.01 a |
| 2nd | 1.189±0.07 a | 0.24±0.01 a |
| 3rd | 1.191±0.11 a | 0.23±0.02 a |
| 4th | 1.196±0.09 a | 0.25±0.02 a |
| 5th | 0.983±0.19 b | 0.41±0.07 b |
| 6th | 0.979±0.24 b | 0.43±0.11 b |
Different letters in the same column indicate a significant difference by ANOVA, p < 0.05.
Conclusions
In this work, recombinant M. gryphiswaldense strains TBC with only one copy of Nb gene in genome and TBC+ containing one copy of Nb gene in genome and multi-copies of pBBR1MCS-2 were constructed. Both strains can produce a novel functional complex of magnetosome-Nbs able to recognize free TBBPA, but the magnetosome-Nbs biosynthesized by TBC+ had higher binding ability to TBBPA than those by TBC. The reusable magnetosome-Nbs from TBC+ showed a promise in the development of an ELISA for the detection of TBBPA in water. Furthermore, it was suggested that TBC+ can be used as a factory to cost-effectively produce eco-friendly Nb-functionalized magnetosomes that have a great prospect in the field of immunochemical technology.
Supplementary Material
Funding information
This work was supported in part by the Key Project of Inter-Governmental International Scientific and Technological Innovation Cooperation (2019YFE0115800), the National Key Research and Development Program of China (2018YFC1602900), the National Institute of Environmental Health Sciences (NIEHS) Superfund Research Program (P42ES04699), NIEHS RIVER Award (R35ES030443), and the USDA Hatch Project (HAW05044-R).
Footnotes
Conflict of interest: The authors declare that they have no conflict of interest.
References
- [1].Raguraman V, Suthindhiran K. Comparative ecotoxicity assessment of magnetosomes and magnetite nanoparticles. Int J Environ Health Res. 2020;30:13–25. 10.1080/09603123.2019.1570489. [DOI] [PubMed] [Google Scholar]
- [2].Ali I, Peng C, Khan ZM, Naz I. Yield cultivation of magnetotactic bacteria and magnetosomes: A review. J Basic Microbiol. 2017;57:643–52. 10.1002/jobm.201700052. [DOI] [PubMed] [Google Scholar]
- [3].He J, Tian J, Xu J, Wang K, Li J, Gee SJ, et al. Strong and oriented conjugation of nanobodies onto magnetosomes for the development of a rapid immunomagnetic assay for the environmental detection of tetrabromobisphenol-A. Anal Bioanal Chem. 2018;410:6633–42. 10.1007/s00216-018-1270-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [4].He J, Ma S, Wu S, Xu J, Tian J, Li J, et al. Construction of immunomagnetic particles with high stability in stringent conditions by site-directed immobilization of multivalent nanobodies onto bacterial magnetic particles for the environmental detection of tetrabromobisphenol-A. Anal Chem. 2020;92:1114–21. 10.1021/acs.analchem.9b04177. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [5].Lang C, Schüler D. Expression of green fluorescent protein fused to magnetosome proteins in microaerophilic magnetotactic bacteria. Appl Environ Microbiol. 2008;74:4944–53. 10.1128/AEM.00231-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [6].Scheffel A, Gärdes A, Grünberg K, Wanner G, Schüler D. The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals. J Bacteriol. 2008;190:377–86. 10.1128/jb.01371-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [7].Ohuchi S, Schüler D. In vivo display of a multisubunit enzyme complex on biogenic magnetic nanoparticles. Appl Environ Microbiol. 2009;75:7734–8. 10.1128/AEM.01640-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [8].Pollithy A, Romer T, Lang C, Müller FD, Helma J, Leonhardt H, et al. Magnetosome expression of functional camelid antibody fragments (nanobodies) in Magnetospirillum gryphiswaldense. Appl Environ Microbiol. 2011;77:6165–71. 10.1128/AEM.05282-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [9].Mickoleit F, Lanzloth C, Schüler D. A versatile toolkit for controllable and highly selective multifunctionalization of bacterial magnetic nanoparticles. Small. 2020;16:1906922. 10.1002/smll.201906922. [DOI] [PubMed] [Google Scholar]
- [10].Nudelman H, Valverde-Tercedor C, Kolusheva S, Perez Gonzalez T, Widdrat M, Grimberg N, et al. Structure–function studies of the magnetite-biomineralizing magnetosome-associated protein MamC. J Struct Biol. 2016;194:244–52. 10.1016/j.jsb.2016.03.001. [DOI] [PubMed] [Google Scholar]
- [11].Duarte JN, Cragnolini JJ, Swee LK, Bilate AM, Bader J, Ingram JR, et al. Generation of immunity against pathogens via single-domain antibody–antigen constructs. J Immunol. 2016;197:4838–47. 10.4049/jimmunol.1600692. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [12].Wu S, Ma F, He J, Li QX, Hammock BD, Tian J, et al. Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1. J Nanobiotechnology. 2021;19:27. 10.1186/s12951-021-00773-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [13].Ladenson RC, Crimmins DL, Landt Y, Ladenson JH. Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment. Anal Chem. 2006;78:4501–8. 10.1021/ac058044j. [DOI] [PubMed] [Google Scholar]
- [14].Kim H-J, McCoy MR, Majkova Z, Dechant JE, Gee SJ, Tabares-da Rosa S, et al. Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay. Anal Chem. 2012;84:1165–71. 10.1021/ac2030255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [15].He T, Wang Y, Li P, Zhang Q, Lei J, Zhang Z, et al. Nanobody-based enzyme immunoassay for aflatoxin in agro-products with high tolerance to cosolvent methanol. Anal Chem. 2014;86:8873–80. 10.1021/ac502390c. [DOI] [PubMed] [Google Scholar]
- [16].Wang J, Bever CRS, Majkova Z, Dechant JE, Yang J, Gee SJ, et al. Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A. Anal Chem. 2014;86:8296–302. 10.1021/ac5017437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [17].Covaci A, Voorspoels S, Abdallah MA-E, Geens T, Harrad S, Law RJ. Analytical and environmental aspects of the flame retardant tetrabromobisphenol-A and its derivatives. J Chromatogr A. 2009;1216:346–63. 10.1016/j.chroma.2008.08.035. [DOI] [PubMed] [Google Scholar]
- [18].Zheng X, Sun R, Qiao L, Guo H, Zheng J, Mai B. Flame retardants on the surface of phones and personal computers. Sci Total Environ. 2017;609:541–5. 10.1016/j.scitotenv.2017.07.202. [DOI] [PubMed] [Google Scholar]
- [19].Hayama T, Yoshida H, Onimaru S, Yonekura S, Kuroki H, Todoroki K, et al. Determination of tetrabromobisphenol A in human serum by liquid chromatography–electrospray ionization tandem mass spectrometry. J Chromatogr B. 2004;809:131–6. 10.1016/j.jchromb.2004.06.013. [DOI] [PubMed] [Google Scholar]
- [20].Okeke ES, Huang B, Mao G, Chen Y, Zhengjia Z, Qian X, et al. Review of the environmental occurrence, analytical techniques, degradation and toxicity of TBBPA and its derivatives. Environ Res. 2022;206:112594. 10.1016/j.envres.2021.112594. [DOI] [PubMed] [Google Scholar]
- [21].Fu H-J, Wang Y, Xiao Z-L, Wang H, Li Z-F, Shen Y-D, et al. A rapid and simple fluorescence enzyme-linked immunosorbent assay for tetrabromobisphenol A in soil samples based on a bifunctional fusion protein. Ecotoxicol Environ Saf. 2020;188:109904. 10.1016/j.ecoenv.2019.109904. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [22].Decherf S, Seugnet I, Fini J-B, Clerget-Froidevaux M-S, Demeneix BA. Disruption of thyroid hormone-dependent hypothalamic set-points by environmental contaminants. Mol Cell Endocrinol. 2010;323:172–82. 10.1016/j.mce.2010.04.010. [DOI] [PubMed] [Google Scholar]
- [23].Viberg H, Eriksson P. Differences in neonatal neurotoxicity of brominated flame retardants, PBDE 99 and TBBPA, in mice. Toxicology. 2011;289:59–65. 10.1016/j.tox.2011.07.010. [DOI] [PubMed] [Google Scholar]
- [24].Rong C, Huang Y, Zhang W, Jiang W, Li Y, Li J. Ferrous iron transport protein B gene (feoB1) plays an accessory role in magnetosome formation in Magnetospirillum gryphiswaldense strain MSR-1. Res Microbiol. 2008;159:530–6. 10.1016/j.resmic.2008.06.005. [DOI] [PubMed] [Google Scholar]
- [25].Zhang Y, Zhang X, Jiang W, Li Y, Li J. Semicontinuous culture of Magnetospirillum gryphiswaldense MSR-1 cells in an autofermentor by nutrient-balanced and isosmotic feeding strategies. Appl Environ Microbiol. 2011;77:5851–6. 10.1128/AEM.05962-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [26].Wang Q, Liu J-X, Zhang W-J, Zhang T-W, Yang J, Li Y. Expression patterns of key iron and oxygen metabolism genes during magnetosome formation in Magnetospirillum gryphiswaldense MSR-1. FEMS Microbiol Lett. 2013;347:163–72. 10.1111/1574-6968.12234. [DOI] [PubMed] [Google Scholar]
- [27].Wang J, Majkova Z, Bever CRS, Yang J, Gee SJ, Li J, et al. One-step immunoassay for tetrabromobisphenol A using a camelid single domain antibody–alkaline phosphatase fusion protein. Anal Chem. 2015;87:4741–8. 10.1021/ac504735p. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [28].Yang Y, Lu L, Zhang J, Yang Y, Wu Y, Shao B. Simultaneous determination of seven bisphenols in environmental water and solid samples by liquid chromatography–electrospray tandem mass spectrometry. J Chromatogr A. 2014;1328:26–34. 10.1016/j.chroma.2013.12.074. [DOI] [PubMed] [Google Scholar]
- [29].Yoon T-J, Lee W, Oh Y-S, Lee J-K. Magnetic nanoparticles as a catalyst vehicle for simple and easy recycling. New J Chem. 2003;27:227–9. 10.1039/B209391J. [DOI] [Google Scholar]
- [30].Bever CS, Dong J-X, Vasylieva N, Barnych B, Cui Y, Xu Z-L, et al. VHH antibodies: emerging reagents for the analysis of environmental chemicals. Anal Bioanal Chem. 2016;408:5985–6002. 10.1007/s00216-016-9585-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [31].Xu T, Wang J, Liu S, Lü C, Shelver WL, Li QX, et al. A highly sensitive and selective immunoassay for the detection of tetrabromobisphenol A in soil and sediment. Anal Chim Acta. 2012;751:119–27. 10.1016/j.aca.2012.06.030. [DOI] [PubMed] [Google Scholar]
- [32].Bu D, Zhuang H, Zhou X, Yang G. Biotin–streptavidin enzyme-linked immunosorbent assay for detecting Tetrabromobisphenol A in electronic waste. Talanta. 2014;120:40–6. 10.1016/j.talanta.2013.11.080. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.