Overview
Key numbers
| Step | Variable | Value |
|---|---|---|
| Library input | Ph.D.-7 phage clones per panning round | 1 × 10¹¹ PFU in 100 µL 0.05% TBST |
| Negative selection | Metal targets immobilised (10 mM each) | Hg(NO3)2 → Cd standard → MnCl2 → NiCl2, sequentially |
| Positive selection | Target metal | 10 mM CuSO4, three rounds |
| Round-3 output | Plaques picked for amplification and sequencing | 240 |
| Sequencing yield | Unique 7-mer peptide sequences | 20 (P-1 through P-20) |
| Top-affinity clones (Fig. 3a, qualitative ELISA PFU/mL means) | P-10 DTAHGTW (1 His); P-11 MHIVPHE (2 His); P-12 DRSWVTS (0 His); P-18 ASKGVGL (0 His); P-20 EARAPGS (0 His) | P-11 selected as the candidate for further work based on its MH (Met-His) motif at the N-terminus and a second histidine at position 6 (paper wording: “two histidine at both ends of the peptide” and “peptides with MH motifs”) |
| Cross-reactivity ELISA (Fig. 4b, MHIVPHE clone, %PFU normalised to Cu = 100%) | Cu²⁺ | 100% reference |
| Cross-reactivity ELISA | Ni²⁺ | ≈45% (large error bar) |
| Cross-reactivity ELISA | Mn²⁺ | ≈38% |
| Cross-reactivity ELISA | Cd²⁺ | ≈18% |
| Cross-reactivity ELISA | Hg²⁺ | ≈10% (nominal; bar within error of zero) |
| DNS-L9 fluorescence-quenching response (Fig. 5b, 1 - F/F0 normalised; 10 µM DNS-L9 + 10 µM metal ion = 1 equiv in 50 mM HEPES pH 7.41, ex 330 nm) | Cu²⁺ | ≈0.58 quenching ratio (≈58.5% quenching) |
| DNS-L9 fluorescence response | Pb²⁺, Hg²⁺, Cd²⁺, Ni²⁺, Mn²⁺, Cr³⁺, As³⁺, Na⁺, K⁺ | Each ≈0 (no significant change vs blank) |
| Statistics | Mann–Whitney U comparison of Cu vs Hg, Cu vs Cd | p ≈ 0.1 (authors state as “approximately 0.1”; with n = 3 independent ELISA replicates the test is underpowered) |
| Cited regulatory anchor | WHO drinking-water guideline for Cu (paper’s wording: “WHO suggests that the concentration of copper in drinking water should be less than 31 µM”) | <31 µM |
| Cited environmental context | Reported industrial wastewater Cu²⁺ | 2.5 to 10,000 mg/L |
Methods (brief)
Phage display library and host. Ph.D.-7 Phage Display Peptide Library Kit (New England Biolabs GmbH, Frankfurt am Main, Germany) screened on Escherichia coli K12 ER 2738 (NEB), grown overnight at 37 °C in LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) with 20 µg/mL tetracycline; agar plates 15 g/L with 20 mg/L tetracycline.
Microplate functionalisation. Maleic anhydride-activated microtiter plates (Thermo Scientific Cat#15100) functionalised overnight at room temperature with 100 µL of 10 mM Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (BCML; Sigma Aldrich Cat# 14580) in 0.1 M NaPO4 pH 8, then washed three times with 300 µL 0.05% TBST, blocked with 3% BSA in 0.05% TBST (2 h, room temperature), washed again, and modified with 20 µL of 10 mM metal salts for 20 min at room temperature. Metal salts used: Hg(NO3)2, Pb(NO3)2, MnCl2, ZnSO4, CuSO4, NiCl2; cadmium (Cd) standard solution, chromium (Cr) standard solution, NaAsO2, KCl, and NaCl (all 10 mM).
Biopanning. Library diluted to 1 × 10¹¹ PFU in 100 µL 0.05% TBST, applied first to a Ni(II)-coated well (negative selection), unbound clones transferred to Hg(II)-coated, Mn(II)-coated, and Cd(II)-coated wells in sequence (text and Figure 2 schematic are consistent on which non-target metals are used; the negative-selection ordering is described in slightly different orderings in two passages — page 3 lists mercury (Hg) → Cd → Mn → nickel (Ni), the Materials-and-Methods Surface Panning section lists negative selection against Ni, Mn, Cd, Hg). Three positive-selection rounds were then performed against Cu(II); 100 µL of 0.2 M glycine-HCl pH 2.2 was used for elution and neutralised with 15 µL of 1 M Tris-HCl pH 9.1 after 15 min incubation. After the third round, 240 plaques were selected from LB/IPTG/X-gal titration plates for DNA sequencing; 20 unique 7-mer copper-binding peptide sequences were identified.
Affinity ELISA. Plates functionalised and modified as above with Cu(II), incubated with 100 µL of 10⁹ PFU per well of each selected clone (n = 21 sequences screened in Fig. 3a; the higher-affinity hits P-10, P-11, P-12, P-18, P-20 were taken forward); plates washed 10× with TBST; phage eluted with 100 µL of 0.2 M glycine-HCl pH 2.2 and titrated per kit manual. Three independent experiments per clone; mean PFU/mL plotted with standard deviation in Fig. 3a.
Cross-reactivity ELISA. Selective P-11 clone tested on plates functionalised with Cu(II), Mn(II), Hg(II), Ni(II), and Cd(II) at 10 mM modification; 100 µL of 10⁹ PFU selected clones per well; phage titre on Cu wells set to 100% reference and other-metal titres normalised. Three independent experiments.
Peptide synthesis. DNS-L9 (Dansyl-Met-His-Ile-Val-Pro-His-Glu-Lys-Trp-NH2) synthesised on Rink Amide resin (0.1 mmol) by microwave-assisted Fmoc SPPS on an Initiator+Alstra automated synthesizer (Biotage, Sweden). Couplings performed twice for 5 min at 75 °C with Fmoc-amino acid (5 equiv), DIC (5 equiv), Oxyma (5 equiv) in DMF. Fmoc deprotection in 20% piperidine/DMF (1×3 min, 1×10 min). Dansyl chloride (5 equiv) and TEA (3 equiv) coupled to peptidyl resin for 4 h. Cleavage with TFA/TIS/H2O 95:2.5:2.5 (2 h); precipitation in cold diethyl ether; lyophilisation. Crude product characterised by reverse-phase HPLC on a Shimadzu Prominence-i LC-2030C Plus with a Jupiter 4 µm Proteo 90 Å, 4.6 × 250 mm column, UV detection at 224 nm, linear gradient 5–95% B over 60 min at 1 mL/min (A = water + 0.1% TFA; B = acetonitrile + 0.1% TFA). ESI(+) mass confirmation on a LCMS 2020 Shimadzu single-quadrupole at 1.5 mL/min isocratic 60% B with 0.1% formic acid eluents.
Fluorescence sensing. DNS-L9 dissolved in double-distilled water at 2 mM and stored at 4 °C. Metal-ion stocks at 10 mM prepared from Hg(NO3)2, Pb(NO3)2, MnCl2, ZnSO4, CuSO4, NiCl2, Cd standard, Cr standard, NaAsO2, KCl, and NaCl. Fluorescence measured on a Synergy H1MG Multimode Microplate Reader (BioTek Instruments, USA) in 96-well F-bottom Greiner Bio-one plates with excitation at 330 nm; F0 (no metal) and F (with metal) intensities reported. Working solution was 10 µM DNS-L9 in 50 mM HEPES buffer pH 7.41; metal ions added at 1.0 equiv (10 µM).
Statistics. Calculations in Excel; nonparametric Mann–Whitney U test in SciPy (Python 3). Authors state the test gave significant differences for Cu vs Hg and Cu vs Cd “assuming p ≈ 0.1”; with n = 3 independent replicates the statistical power is limited and this caveat is acknowledged in the Statistical analysis section.
Update history
The five most recent substantive edits to this page, classified major (evidence or structure moved), correction (a published value or statement was wrong and has been fixed), or minor (narrative rewritten without changing the underlying evidence). Each description is derived from what the edit did to this page; the linked commit is the authoritative record, routine regeneration passes are excluded, and the full version history lives in git.
| Commit | Date | Change | Description |
|---|---|---|---|
| b01ec52c31 | 2026-08-04 | major | 1 section added |
| 3171d069e3 | 2026-08-02 | major | 1 section added |
| 90a2f9a38e | 2026-07-30 | major | 1 section added and 1 removed; narrative text revised |
| eaa8eeeedf | 2026-07-27 | major | 1 section added and 1 removed |
| 50de6ab217 | 2026-07-27 | minor | Narrative text revised |