Environmental pollution by various industrial chemicals and biological agents poses serious risks to human health. Especially, marine contamination by PTE has become a global concern in recent years [1
]. Many efforts have been conducted to monitor PTE contaminants in the aquatic environment, in order to gauge the damage and trace the source of contamination, which has primarily involved the direct chemical analysis of aquatic samples [3
]. However, chemical analysis may be insufficient for evaluating the impact of the pollutant on the aquatic organisms. In assessing the risks of environmental pollutants, it is important to understand the damages done to organisms, such as alterations in molecular, cellular, and physiological processes, occurring within an organism, as outcomes of pollutant exposure [5
]. Therefore, the measurement of biomarkers of heavy metal exposure may offer an alternative to conventional chemical analysis. Changes in biomarkers of exposure can provide quantitative as well as qualitative estimates of exposure to various contaminants [8
The induction of metallothionein (MT) synthesis by heavy metals has been demonstrated in numerous species (e.g., mollusks, crustaceans, annelids), prompting suggestions of MT concentrations in organisms as potential biomarkers of heavy metal exposure [10
]. MTs are low-molecular-weight range proteins of 6–7 kDa with unusually high (20–30%) cysteine contents and involved in metal detoxification and homeostasis in living organisms, by coordinating to metal ions with high affinity. In the previous study, significantly higher mRNA levels of OjaMT
were detected in the liver tissue of O. javanicus
after 24 h exposure of heavy metals at 0.1–100 μg/mL level [12
Traditionally, the expression levels of protein biomarkers have been determined indirectly by measuring changes in messenger RNA (mRNA) levels [13
]. However, many studies have reported that the correlation between mRNA and protein expression levels is relatively inconsistent [14
]. Alternatively, a few groups have reported the use of enzyme-linked immunosorbent assay (ELISA) for quantitative analysis of MT protein [17
]. These studies have shown that it is important to quantify protein biomarkers directly to obtain more relevant information. Label-free biosensors are particularly useful tools for the quick detection in crude samples. The surface plasmon resonance (SPR)-based biosensor is a representative label-free technique, capable of detecting low concentrations of biomaterials within a rapid response time [19
In this paper, we developed an approach to monitor the heavy metal contamination of marine ecosystems via detection of MT proteins using SPR. We fabricated sensor chips tailored to present anti-Oryzias javanicus MT (anti-OjaMT) single-chain fragment of variable region (scFv) or anti-OjaMT monoclonal antibody (mAb) and evaluated the performance of these sensor chips in OjaMT detection. We also attempted to establish a platform enabling point-of-care testing (POCT) by utilizing portable SPR.
2. Materials and Methods
2.1. General Procedures
A plasmid DNA Mini-Prep kit and all restriction enzymes were purchased from New England Biolabs (Beverly, MA, USA) and Elpis Biotech (Daejeon, Korea). The polymerase chain reaction (PCR) kit and gel extraction product kit came from Real Biotech Corp (Taipei, Taiwan). All oligomers were commercially synthesized by Bioneer (Daejeon, Korea). General chemicals were product of Sigma Aldrich (St. Louis, MO, USA) in the best grade available. The scFv-Cys3 gene was cloned in the bacterial expression vector pET-28a(+). We used this pET-28a(+)-scFv-Cys3 construct to express a recombinant scFv-Cys3 in Escherichia coli. The expression vector and Rosetta (DE3) were purchased from Novagen (Madison, WI, USA). Isopropyl-β-d-thiogalactopyranoside (IPTG) was obtained from Gold Biotechnology (St. Louis, MO, USA). Protein samples were analyzed on 12% polyacrylamide gels and stained with Coomassie Brilliant Blue R250. The gold chip was procured from MiCo NanoBioSys. SPR was performed using a MiCo SPR nano-instrument from MiCo NanoBioSys, using the gold sensor chip.
2.2. Purification of Anti-OjaMT mAbs
The mAbs in serum-free hybridoma supernatants were passed through a 0.22 µm filter and applied to a Protein G Sepharose column purchased from Abcam (Cambridge, UK). After washing with 1× PBS, the bound mAb was eluted with 0.2 M glycine, pH 2.0, neutralized with 1 M Tris, pH 8.0, and dialyzed against 1× PBS containing 0.02% (w/v) sodium azide. The purity of the mAb preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using a 12% SDS-PAGE gel.
2.3. Expression and Purification of Recombinant OjaMT
Rosetta (DE3) cells were transformed with OjaMT cloned in pRSET-C [12
]. The transformants were grown to an OD600 nm of 0.6 at 37 °C in shake flasks containing 200 mL of Luria-Bertani (LB) medium (0.5% yeast extract, 1.0% tryptophan, and 1.0% NaCl) with 50 µg/mL ampicillin. The expression was induced by adding IPTG to a final concentration of 0.3 mM. The transformed cells were further grown overnight at 18 °C. The cells were harvested by centrifugation at 3000 rpm for 30 min. The cell pellets were re-suspended in lysis buffer (50 mM NaH2
, 300 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF) at pH 8.0) and lysed by sonication. The solubilized protein was separated by centrifugation at 15,000 rpm/4 °C for 30 min. The supernatant was incubated with Ni-NTA resin at 4 °C for 2 h and then applied to the column. The His-tagged recombinant OjaMTs were eluted with native elution buffer (50 mM NaH2
, 300 mM NaCl, and 250 mM imidazole at pH 8.0). The eluted recombinant OjaMTs were dialyzed against PBS at 4 °C to remove imidazole.
2.4. Immunoblot Analysis
Purified OjaMT was separated by SDS-PAGE and transferred to a 0.45 µm polyvinylidene fluoride (PVDF) membrane purchased from Millipore (Burlington, MA, USA). This was blocked with 10% (w/v) skim milk powder in TBS, and the immobilized proteins were detected with anti-OjaMT mAb and a secondary goat anti-mouse IgG HRP conjugate obtained from Bethyl (Montgomery, AL, USA), followed by detection with PicoEPD Western Reagent purchased from Elpis Biotech (Daejeon, Korea).
2.5. Isolation of the Variable Heavy and Light Chain (VH and VL)
Total RNA was extracted from the OjaMT hybridoma cells (Youngin Frontier, Seoul, Korea) using TRI Reagent®. RNA (1 µg) was treated with DNaseΙ and reverse-transcribed into complementary DNA (cDNA) for use as a template for PCR. The reverse transcription was performed at 37 °C for 2 h in a reaction volume of 20 µL containing the following: 1 µL of specific primer (80 µM), 10× Maloney murine leukemia virus (M-MLV) reverse transcription buffer, sterile water (RNase-free, 14 µL), deoxyribonucleotide triphosphates (dNTPs) (2 mM each), RNase inhibitor (40 units/µL), and M-MLV reverse transcriptase (200 units/µL). Reverse transcriptase activity was stopped by heating samples at 72 °C for 10 min. Heavy-chain cDNA was obtained using MuIgG3-Fwd (heavy chain) primer, 5′-CTG GAC AGG GCT CCA TAG TTC CA-3′, and light chain cDNA was obtained MuCK-Fwd (light chain) primer: 5′-CTC ATT CCT GTT GAA GCT CTT GAC-3′. The resulting cDNA was subjected to PCR using the primer set of MuJH1For, 5′-TGA GGA GAC GGT GAC CGT GGT CCC-3′, and MuVH4/6Back, 5′-GAG GTY CAG CTG CAR CAR TCT GG-3′, to amplify the VH gene and the primer set of MuJK1For, 5′-TTT GAT TTC CAG CTT GGT GCC TCC-3′, and MuVK1Back, 5′-GAC ATT GTG ATG WCA CAG TCT CC-3′, to amplify VL gene.
2.6. Construction of Cys3-Tagged scFv (scFv-Cys3)
A linker1 and linker2 gene coding (GGGGS)3
(GS-linker) were prepared by PCR amplification of GS-linker gene (GGT GGC GGT GGC TCG GGC GGT GGT GGG TCG GGT GGC GGC GGA TCT) by using the primer sets of LinkFor,5′-AGA TCC GCC GCC AC-3′, RevMuJH1For-2, 5′-CGG TCA CCG TCT CCT CAG GTG GCG GTG GCT C-3′, RevMuVK2 Back-2, 5′-CTG TTG CAG CTG AAC CTC AGA TCC GCC GCC AC-3′, and LinkRev, which was 5′-GGT GGC GGT GGC T-3′, respectively. The linker1 gene was annealed to VH
and the annealed gene product was used as a template for PCR amplification using a primer set of MuJK1For, 5′-TTT GAT TTC CAG CTT GGT GCC TCC-3′, and LinkRev, 5′-GGT GGC GGT GGC T-3′, to produce VH
-linker (Figure 1
a). The linker2 gene was annealed to VL
fragments and the annealed gene product was used as a template for PCR amplification using a primer set of LinkFor, 5′-AGA TCC GCC GCC AC-3′, and MuVH4/6Rev, 5′-GAG GTY CAG GTC CAR CAR TCT GG-3′, to generate linker-VL
In the final assembly reaction, the VH-linker and linker-VL genes were annealed with each other and the annealed product was used as a template for jumping PCR using a primer set of VH, (Nde I)-F, 5′-CAT ATG GAG GTT CAG CTG CAG CAG T-3′, and VL, (Hind III)-R, 5′-AAG CTT TTG ATT TCC AGC TTG GTG CCT-3′, to generate scFv. The recombinant scFv fragment (VH-linker-VL) was then inserted into a bacterial expression vector pET-28a(+) using Nde I/Hind III restriction sites to make pJDH033. The GS-linker-Cys3 fragment was prepared by complementary oligonucleotide synthesis to generate the sequence: 5′-AGC TTG GTG GCG GTG GCT CGG GCG GTG GTG GGT CGG GTG GCG GCG GAT CTT GTT GCT GTT GAC-3′. This double strand DNA was inserted into the Hind III/Xho I sites of pJDH033 to produce pJDH043.
2.7. Expression and Purification of Recombinant scFv
E. coli Rosetta (DE3) cells were transformed with pJDH043. The transformants were grown to an OD600 nm of 0.6 at 37 °C in shake flasks containing 200 mL of LB medium with 50 µg/mL kanamycin. The expression was induced by adding IPTG to a final concentration of 0.3 mM. The transformed cells were further grown overnight at 18 °C. The cells were harvested by centrifugation at 3000 rpm for 30 min. The cell pellets were re-suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF at pH 8.0) and lysed by sonication. The soluble and insoluble fractions were then separated by centrifugation at 15,000 rpm/4 °C for 30 min. Lysis buffer containing 8 M urea was added to solubilize the pellet, and the solution was incubated with Ni-NTA resin at 4 °C, with stirring for 3 h. The loaded column was washed stepwise with 50 mL of 4, 3, 2, 1, and 0.5 M urea, and finally with 50 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole at pH 8.0). The His6-tagged recombinant scFv’s were eluted with native elution buffer (50 mM NaH2PO4, 300 mM NaCl, and 250 mM imidazole at pH 8.0). The eluted recombinant scFv’s were dialyzed against PBS at 4 °C to remove imidazole. Protein samples were analyzed by denaturing SDS-PAGE. The gel was dyed with Coomassie Blue staining solution. The protein concentration was determined by the Bradford method, with BSA as the standard.
2.8. Experimental Fish
We used 6–9-month-old Javanese medaka (Oryzias javanicus, 2.4–2.8 mm; weight: 0.13–0.17 g) which were cultured in the KIOST (Geoje, Korea). Twenty fish were placed in a 3 L tank at 25 ± 1 °C in natural seawater filtered through serially connected filters of different pore sizes (100, 10, and 1 μm) with a controlled normal photo-regimen (16 h light–8 h dark). The fish were fed with Artemia sp. naupii once a day.
2.9. CdCl2 Exposure and Sampling (Heavy Metal Exposure and Sampling)
Four groups of five fishes were prepared. The fish were transferred to 1 L beakers containing 0.8 L of seawater. After acclimation for 48 h without food, the experimental groups were exposed to 0.1, 1, and 10 mg/L CdCl2
(Sigma-Aldrich Ltd.) for 24 h, respectively. The exposure concentrations were selected after consideration of the 24 h LC50
(the median lethal concentrations) value for Javanese medaka (44.25 mg/L) [21
]. A group of untreated fish was prepared as a control. Liver tissues were collected from each group after rendering fish unconscious with cold shock. Total protein was extracted from the fish livers with Pro-Prep Protein Extraction Solution (Intron Biotechnology, Sungnam, Korea), according to the manufacturer’s instructions.
2.10. SPR Analysis
The gold chip surface was cleaned with concentrated piranha solution (H2SO4:H2O2 = 7:3 v/v) and thoroughly rinsed with ethanol and deionized water. All SPR spectroscopy experiments were performed with the pre-cleaned gold chip on a MiCo SPR nano-device at room temperature using the PBS buffer as a running solution unless indicated otherwise. The SPR sensor chip presenting antibodies was prepared by coating the protein G-Cys3 (0.1 mg/mL, 10 µL/min for 15 min) onto the gold substrate and capturing the anti-OjaMT mAb site-specifically (50 µg/mL, at a flow rate of 10 µL/min for 15 min). Unconjugated antibody was washed and blocked by flowing PBS and PEG–thiol solution (0.1 mM, 10 µL/min for 10 min), sequentially. The SPR sensor chip presenting scFv was prepared by coating the scFv-Cys3 (50 µg/mL, 10 µL/min for 15 min) onto the gold substrate. The scFv-presenting sensor chip was washed with PBS and blocked with PEG–thiol solution (0.1 mM, 10 µL/min for 10 min). After the immobilization of probes, the OjaMT (5–30 µg/mL, in PBS) was flowed over the sensor chip at 10 µL/min for 10 min. The sensor chip was washed with PBS to remove unbound OjaMT, and the difference in RU was recorded.
In this paper, we described an approach to monitor the heavy metal exposure of the marine organism Oryzias javanicus, by measuring a biomarker protein, metallothionein, in real time. The approach is based on a portable SPR system, which allows label-free detection of target proteins. For this work, we prepared two types of detection probes, namely, anti-OjaMT monoclonal antibody and anti-OjaMT scFv. The anti-OjaMT monoclonal antibody was harvested using hybridoma cells, and scFv was recombinantly prepared based on a jumping PCR assembly method. The recombinant scFv was modified to carry Cys3-tag for site-directed immobilization. The sensor chips were made by immobilizing anti-OjaMT monoclonal antibody or anti-OjaMT scFv and were tested for in situ label-free OjaMT detection. The scFv-presenting sensor chips showed improved performance compared to antibody-presenting sensor chips. The sensor chip was fabricated by short immobilization steps, without the presence of the large linker protein, protein G, which can mediate unwanted interactions. The scFv sensor chips were finally used for the detection of OjaMT in fish liver from heavy metal contaminated Oryzias javanicus. We were able to distinguish the contaminated samples from non-treated control. The developed system provides a generic platform to monitor target biomarkers in real time for environmental contamination monitoring.