Sosnowska et al. 2025 — Phage-display-derived peptide chemosensor for Cu(II) in aquatic samples

Sosnowska, Łęga, Olszewski, and Gromadzka (Institute of Biotechnology and Molecular Medicine, Gdańsk; Faculty of Chemistry, Warsaw University of Technology; Nano Expo Sp. z o.o., Gdańsk) report a methodology paper that combines phage display biopanning, peptide synthesis, and fluorescence spectroscopy to develop a Cu(II)-selective fluorescent chemosensor. The motivation is sensor development for ecotoxicological monitoring of copper in aquatic ecosystems; the WHO drinking-water reference cited in the paper is <31 µM total copper and reported industrial-wastewater Cu²⁺ concentrations span 2.5 to 10,000 mg/L. The biopanning workflow modifies the standard Ph.D.-7 protocol by introducing four sequential negative-selection rounds (Hg²⁺ → Cd²⁺ → Mn²⁺ → Ni²⁺) prior to three positive rounds against Cu²⁺, which the authors state is the first reported use of microtiter-plate functionalisation chemistry to enable negative selection against multiple non-target metals. The selected 7-mer MHIVPHE (Met-His motif at the N-terminus and a second histidine near the C-terminus at position 6 of the 7-residue peptide; the paper frames this as “two histidines at both ends” with an N-terminal MH motif) was extended into a dansyl-labelled decapeptide DNS-L9 (Dansyl-Met-His-Ile-Val-Pro-His-Glu-Lys-Trp-NH2) that exhibits ≈58.5% Cu²⁺-induced fluorescence quenching at a 1:1 peptide:metal stoichiometry with negligible response to nine other monovalent and divalent ions tested at the same concentration. Copper is not among the ten HMI HMTc analytes (Pb, tAs, Cd, MeHg, tHg, iAs, Ni, Al, Cr-VI, Sn) and the paper reports no contamination measurements in any food, personal-care, or environmental sample. The source enters the HMI corpus as upstream sensor-development methodology context adjacent to the broader phage-display and peptide-metal-binding literature already represented in the same Kimi peptide folder (bae2000-synthetic-phytochelatin-bacterial-cd-bioaccumulation, hu2024-engineered-ecoli-hg-cr-adsorption, juskowiak2008-cysteine-rich-oboc-peptide-libraries, luo2024-peptides-heavy-metal-remediation, lu2022-metal-chelating-peptides-review, han2025-peptide-zinc-complexation-aquatic-review).

Key numbers

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; Cd standard solution, 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 Hg → Cd → Mn → 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.

Implications

The MHIVPHE peptide and its DNS-L9 chemosensor derivative are reported as a single proof-of-concept against 10 µM reference Cu²⁺ in HEPES buffer, not yet tested in any real environmental, drinking-water, or food matrix. The cited WHO drinking-water guideline <31 µM is the authors’ framing of the regulatory anchor for downstream sensor deployment; the paper does not measure copper in any actual drinking water sample, does not address ICP-MS or AAS comparability, and does not characterise matrix effects from dissolved organic matter, competing cations at environmental ratios (Ca²⁺, Mg²⁺, Fe²⁺, Zn²⁺), or chloride/sulfate interferences. As an HMI source page this is upstream methodology context for the broader peptide-based sensing and heavy-metal-binding peptide literature; it does not contribute occurrence data to any HMI metals or ingredient page, does not constrain any HMTc per-row standard, and does not warrant a Part 9 synthesis trigger on any cell of the contamination index. Copper is not among the ten HMTc analytes, so metals: is empty even though Cu, Hg, Cd, Pb, Ni, Mn, Cr, and As appear throughout the paper as detection-method targets and counter-selection ions.

Verification notes

• PDF read in full (11 pages) via the Read tool: title page, abstract, background (page 2), results including Figs. 1–4 and the P-1…P-20 peptide-sequence table (pages 3–6), DNS-L9 fluorescence panel Fig. 5 (page 7), discussion (page 6–7), conclusion and Materials-and-Methods (pages 8–9), and the full 52-entry reference list (pages 10–11) were all reviewed. Image tool calls (pages 1–6 and 7–11) returned the rendered images of all 11 pages.
• DOI 10.1186/s12934-024-02553-4, raw handle MFK_53-phage-display-technology-in-ecotoxicology-pepti, and cite-key sosnowska2025-phage-display-peptide-copper-chemosensor checked against wiki/sources/ (grep -l for DOI, raw_handle, and author/year cite-key stem); no existing page on any of the three identity dimensions.
• Open access under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 (CC-BY-NC-ND 4.0) per the first-page rights notice; recorded as license: cc-by-nc-nd-4.0. Note this is more restrictive than the CC-BY-4.0 of han2025-peptide-zinc-complexation-aquatic-review and CC-BY-4.0 typical of BMC/Microbial Cell Factories; the NoDerivatives clause prevents redistribution of adapted material but does not affect citation and quotation under fair use, and does not change the page’s role in the corpus.
• Metals discussed in the paper are copper (primary target), with mercury, cadmium, lead, nickel, manganese, chromium, and arsenic appearing in the negative-selection workflow and the DNS-L9 cross-reactivity panel. Copper is not among the ten HMI HMTc analytes (Pb, tAs, Cd, MeHg, tHg, iAs, Ni, Al, Cr-VI, Sn); the other metals appear only as detection-method context, not as occurrence measurements in any matrix. metals: [] is therefore correct under the same convention used for juskowiak2008-cysteine-rich-oboc-peptide-libraries and han2025-peptide-zinc-complexation-aquatic-review.
• No ingredient or product is measured for contamination; ingredients: [] and products: []. The phrase “aquatic samples” in the title refers to the intended downstream application of the chemosensor (drinking water, surface water, industrial wastewater) and is not a sampling-frame description in this study. matrices: [synthetic-peptide-library, methodology-context] records the actual experimental matrix.
• jurisdictions: [GLOBAL] because the work is methodology with no jurisdiction-specific occurrence claims; corresponding author at Institute of Biotechnology and Molecular Medicine (Gdańsk, Poland) and Nano Expo Sp. z o.o. (Gdańsk, Poland); funding via Warsaw University of Technology and Poland’s National Centre for Research and Development (TECHMATSTRATEG-III/0042/2019-00, acronym ASTACUS). The WHO drinking-water reference is cited as an international anchor, not as a Polish regulatory threshold.
• Brand firewall: instrument vendors and reagent catalogue identifiers are scientific-method vendor names and reference materials (NEB Ph.D.-7 kit, Thermo Scientific Cat#15100, Sigma Aldrich Cat# 14580, Biotage Initiator+Alstra, Shimadzu Prominence-i LC-2030C Plus, Shimadzu LCMS 2020, BioTek Synergy H1MG, Greiner Bio-one F-bottom plates) per the Part 12 Exception 2 carve-out for scientific reproducibility; preserved in Methods. No brand names attribute contamination values to any sampled product, so Part 12 is not triggered.
• Wiki/HMTc firewall: no HMTc threshold proposals, no consumer translations, no synthesis claims about the literature consensus on copper toxicity. The WHO <31 µM drinking-water guideline is reported as the paper’s stated regulatory anchor, not as a wiki recommendation.
• Negative-selection ordering. The text on page 3 reports the negative-selection sequence as Ni(II) → Hg(II) → Mn(II) → Cd(II) (“The first microplate was coated with Ni (II) ions… transferred to a second microplate that was subsequently coated with Hg (II) ions. Microplates modified with Mn (II) and Cd (II) were used to propagate two additional negative selection steps”). The Materials-and-Methods section on page 8 lists “Nickel, manganese, cadmium, and mercury were subjected to negative selection” — Ni(II) → Mn(II) → Cd(II) → Hg(II). The two passages are consistent on which four metals were used and on the count of negative-selection rounds; they differ on the listed sequence (Hg position 2 vs position 4). The Methods section above follows the page-3 ordering, which is the more procedural description; the within-paper variation is noted rather than reconciled because the source itself does not resolve it.
• Audit subagent (2026-06-08) flagged two paraphrase drifts in the original draft: (a) framing the MHIVPHE motif as “His-Met-His at the N- and C-flanks” when the paper itself uses “MH (Met-His) motif” and “two histidines at both ends of the peptide” — corrected to match the paper’s framing in both the body prose and the Key numbers table; (b) the original verification note stated page 3 listed the negative-selection order as Hg → Cd → Mn → Ni when the page-3 text actually reads Ni → Hg → Mn → Cd — corrected here. Independent re-verification against pages 3, 5, 7, and 8 of the PDF confirmed both findings.
• Evidence tier: C (single-laboratory primary methodology study with n = 3 independent replicates per ELISA condition, no inter-laboratory replication, no environmental or food-matrix validation, no orthogonal analytical confirmation against ICP-MS or AAS).
• Adjacent context: see bae2000-synthetic-phytochelatin-bacterial-cd-bioaccumulation and hu2024-engineered-ecoli-hg-cr-adsorption for parallel E. coli-displayed metal-binding-peptide bioengineering, juskowiak2008-cysteine-rich-oboc-peptide-libraries for upstream OBOC library methodology, and han2025-peptide-zinc-complexation-aquatic-review / lu2022-metal-chelating-peptides-review for peptide-metal coordination chemistry reviews in the same Kimi peptide folder.

Page history

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