When Metal Complexes Evolve, and a Minor Species Is the Most Active: the Case of Bis(Phenanthroline)Copper in the Catalysis of Glutathione Oxidation and Hydroxyl Radical Generation.

Several copper-ligands, including 1,10-phenanthroline (Phen), have been investigated for anticancer purposes based on their capacity to bind excess copper (Cu) in cancer tissues and form redox active complexes able to catalyse the formation of reactive oxygen species (ROS), ultimately leading to oxidative stress and cell death. Glutathione (GSH) is a critical compound as it is highly concentrated intracellularly and can reduce and dissociate copper(II) from the ligand forming poorly redox-active copper(I)-thiolate clusters. Here we report that Cu-Phen2  speciation evolves in physiologically relevant GSH concentrations. Experimental and computational experiments suggest that at pH 7.4 mostly copper(I)-GSH clusters are formed, but a minor species of copper(I) bound to one Phen and forming ternary complexes with GSH (GS-Cu-Phen) is the redox active species, oxidizing quite efficiently GSH to GSSG and forming HO• radicals. This minor active species becomes more populated at lower pH, such as typical lysosomal pH 5, resulting in faster GSH oxidation and HO• production. Consistently, cell culture studies showed lower toxicity of Cu-Phen2 upon inhibition of lysosomal acidification. Overall, this study underscores that sub-cellular localisation can considerably influence the speciation of Cu-based drugs and that minor species can be the most redox- and biologically- active.

Integrated Design of Homogeneous/Heterogeneous Copper Complex Catalysts to Enable Synergistic Effects on Sulfur and Lithium Evolution Reactions.

Fatal polysulfide shuttling, sluggish sulfur redox kinetics and detrimental lithium dendrites have curtailed the real discharge capacity, working lifespan and safety of lithium-sulfur (Li-S) batteries. Organic small molecule promotors as one type of emerging active catalysts can fulfil the management of the electrochemical species evolution behaviors. Herein, an integrated engineering is organized by synthesizing dual chlorine-bridge enabled binuclear copper complex (Cu2(phen)2Cl2) and its derivative generated in electrolyte (Cu-ETL) as the heterogeneous and homogeneous catalyst, respectively. The well-designed Cu-ETL with a optimized concentration of 0.25 wt.% as a homogeneous enabler offers highly utilized Cu centers and the sufficient interface contact for guiding the Li2S nucleation/decomposition reactions. The Cu2(phen)2Cl2 loaded on carbon spheres as an interlayer (Cu-INT) can break through the catalytic limitation resulting from the saturated concentration of Cu-ETL and thus offers an extended manipulation effect. Benefiting from the synergistic effect, the Li-S battery shows stable cycling at 3 C upon 500 cycles with a capacity degradation rate as low as 0.029% per cycle. Of specific note, an actual cell energy density of 372.1 Wh kg-1 is harvested by a 1.2 Ah-level soft-packaged pouch cell, implying a chance for requiring the demand of high-energy batteries.

Novel highly selective quinoline-based fluorescent chemosensors for quantitative analysis of Cu(II) ion in water and food.

While copper (Cu2+) is a vital cofactor in numerous enzymatic processes, its homeostasis is critical. Selective sensors for Cu2+ in food matrices are paramount for ensuring adherence to safety regulations and dietary interaction studies. In this work, novel derivatives of 8-aminoquinoline (L1-L4) with extended π-conjugation and various N-substituents were synthesized and evaluated as fluorescent sensors for Cu2+. The 2-pyridinecarbonyl-substituted derivative L3 exhibited sharp fluorescence quenching selectively in the presence of Cu2+. This compound presents high selectivity for Cu2+ even in the presence of other metal ions. The L3-based fluorescent sensor provides a Cu2+ detection limit of 77 nM, surpassing many existing sensors. The quantifications of Cu2+ in water, food supplements, and wines using this sensor have demonstrated good agreement with those obtained using the standard ICP technique. Notably, L3 also facilitates Cu2+ detection in microliter sample volumes at subnanomole levels using paper-based sensors, opening doors for portable and cost-effective on-site testing.

Invasiveness modulation of glioma cells by copper complex-loaded nanoarchitectures.

Among the tumors with the highest lethality, gliomas are primary brain tumors associated with common recurrence inclined to metastasize along the neuraxis and occasionally out of the central nervous system. Even though metastasis is the main responsible for death in oncological patients, few dedicated treatments are approved. Therefore, the establishment of effective anti-metastasis agents is the final frontier in cancer research. Interestingly, some copper complexes have demonstrated promising efficacy as antimetastatic agents, but they may cause off-site effects such as the alteration of copper homeostasis in healthy tissues. Thus, the incorporation of copper-based antimetastatic agents in rationally designed nano-architectures can increase the treatment localization reducing the side effects. Here, copper complex loaded hybrid nano-architectures (CuLNAs) are presented and employed to assess the impact of an intracellular copper source on glioma cell invasiveness. The novel CuLNAs are fully characterized and exploited for cell migration modulation in a glioma cell line. The results demonstrate that CuLNAs significantly reduce cell migration without impairing cell proliferation compared to standard gold and copper NAs. A concomitant antimigratory-like regulation of the epithelial-to-mesenchymal transition genes confirmed these results, as the gene encoding for the epithelial protein E-cadherin was upregulated and the other explored mesenchymal genes were downregulated. These findings, together with the intrinsic behaviors of NAs, demonstrate that the inclusion of metal complexes in the nano-architectures is a promising approach for the composition of a family of agents with antimetastatic activity.

Copper(II) Methacrylate Complexes with Imidazole Derivatives-Structural, Spectral and Antitumor Features.

A series of five novel copper(II) complexes with imidazole derivatives having general core Cu(R-Im)2(Macr)2 (Macr = methacrylate anion; R-Im = 2-methylimidazole/2-MeIm, 4-methylimidazole/4-MeIm, 2-ethylimidazole/2-EtIm, 2-isopropylimidazole/2-iPrIm) has been synthesized and characterized by elemental analysis, Fourier Transform Infrared spectroscopy (FTIR), electronic reflectance spectroscopy, cyclic voltammetry, thermal analysis and single crystal X-ray diffraction. All complexes crystalize in a monoclinic crystal system and form a complex supramolecular network developed through hydrogen bonds. The stereochemistry of the copper ion is distorted octahedral except for the compound with 4-methylimidazole for which the geometry is square-pyramidal. The imidazole derivatives act as unidentate while methacrylate ions are chelated except for compound with 4-methylimidazole where is unidentate. All ligands and complexes inhibited B16 murine melanoma cells in a micromolar range, but the complex with 2-isopropylimidazole was more active. Furthermore, all species do not affect the healthy BJ cells in the concentration range used for assays.

Novel copper complex inhibits the proteasome in skin squamous cell carcinoma induced by DMBA in mice.

The proteasomal system is becoming a target for the treatment of several diseases, especially in cancer therapy. The present study aims to develop a novel copper complex that inhibits the proteasome in skin squamous cell carcinoma. New molecules based on the copper complex were synthesized for the first time to assess their potential as proteasome inhibitors, specifically targeting squamous cell carcinoma induced by 7,12-dimethylbenz(a)anthracene (DMBA) in mouse models. Fourier Transform Infrared (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), nuclear magnetic resonance (NMR), and energy dispersive X-ray analysis (EDX) were carried out to characterize this new copper complex. Notably, the presence of a papilloma (skin tumor) was confirmed by histopathological analysis. Subsequent investigation included the quantification of proteasome levels using a sandwich ELISA test, and the catalytic activity of the 20S proteasome was determined by measuring the fluorescence emitted after the cleavage of 7-amino-4-methylcoumarin (AMC). Hence, X-ray crystallography indicates that all Cu atoms are five-coordinated in a square-pyramidal configuration and biological activity of copper Schiff base complex, which exhibits high proteasome inhibitory activities with particular selectivity of ß5 subunit. The pharmacokinetic properties (ADMET) of the copper complex named Cu(L1) showed encouraging results with very low toxicity, distribution, and absorption. Structure-activity relationship (SAR) information obtained from Cu(L1) demonstrated its selectivity and potent inhibition for ß5 subunit. In this regard, this copper complex has emerged as a novel therapy for skin cancer.

Thiazole Functionalization of Thiosemicarbazone for Cu(II) Complexation: Moving toward Highly Efficient Anticancer Drugs with Promising Oral Bioavailability.

In this work, we report the synthesis of a new thiosemicarbazone-based drug of N'-(di(pyridin-2-yl)methylene)-4-(thiazol-2-yl)piperazine-1-carbothiohydrazide (HL) featuring a thiazole spectator for efficient coordination with Cu(II) to give [CuCl(L)]2 (1) and [Cu(NO3)(L)]2 (2). Both 1 and 2 exhibit dimeric structures ascribed to the presence of di-2-pyridylketone moieties that demonstrate dual functions of chelation and intermolecular bridging. HL, 1, and 2 are highly toxic against hepatocellular carcinoma cell lines Hep-G2, PLC/PRF/5, and HuH-7 with half maximal inhibitory concentration (IC50) values as low as 3.26 nmol/mL (HL), 2.18 nmol/mL (1), and 2.54 × 10-5 nmol/mL (2) for PLC/PRF/5. While the free ligand HL may elicit its anticancer effect via the sequestration of bio-relevant metal ions (i.e., Fe3+ and Cu2+), 1 and 2 are also capable of generating cytotoxic reactive oxygen species (ROS) to inhibit cancer cell proliferation. Our preliminary pharmacokinetic studies revealed that oral administration (per os, PO) of HL has a significantly longer half-life t1/2 of 21.61 ± 9.4 h, nearly doubled as compared with that of the intravenous (i.v.) administration of 11.88 ± 1.66 h, certifying HL as an effective chemotherapeutic drug via PO administration.

Cellular Effects of Cationic Copper(II) Schiff Base Complexes: Anti-Inflammatory and Antiproliferative Properties.

A series of potassium isothiocyanato-(N-salicylidene-aminoacidato) cuprates (1-5) with the general formula of the monomeric unit K[Cu(sal-aa)(NCS)] ⋅ xH2O (x=0 or 2), containing a Schiff-base ligand (H2sal-aa) derived from natural amino acids such as glycine, DL-α-alanine, DL-valine, DL-phenylalanine and ß-alanine, and salicylaldehyde, was screened for in vitro antiradical and major cellular effects against selected cancerous and normal cells. The complexes exhibited strong antioxidant properties against superoxide in vitro and a protective effect on DNA under Fenton-like reaction conditions. Screening of their cellular effects revealed moderate in vitro cytotoxicity against human cancer cell lines (A2780, A2780R and MCF-7), with IC50 values of 25-35 µM, and relatively low toxicity to normal fibroblast MRC-5 cells (with IC50 values>50 µM). Additional experiments performed on A2780 cells revealed that the most potent complex 5 significantly increased the number of A2780 cells arrested in the G2/M phase of the cell cycle and triggered intracellular oxidative stress. The selected flow cytometry experiments (detection of apoptosis/autophagy and activation of caspases 3/7 and depletion of mitochondrial membrane potential) did not reveal the dominant mechanism underlying the cytotoxicity of the complexes but clearly differentiated their molecular effects from those of the reference drug cisplatin. All the complexes exerted anti-inflammatory effects by modulating the levels of the proinflammatory cytokines TNF-α and IL-1ß in LPS-activated THP-1 macrophage-like cells. Complex 5 also slightly influenced the activity of the upstream NF-κB transcription factor, while no effect on PPARγ activation was detected.

An 8-aminoquinoline-naphthyl copper complex causes apoptotic cell death by modulating the expression of apoptotic regulatory proteins in breast cancer cells.

Breast cancer is one of the most common cancers globally and a leading cause of cancer-related deaths among women. Despite the combination of chemotherapy with targeted therapy, including monoclonal antibodies and kinase inhibitors, drug resistance and treatment failure remain a common occurrence. Copper, complexed to various organic ligands, has gained attention as potential chemotherapeutic agents due to its perceived decreased toxicity to normal cells. The cytotoxic efficacy and the mechanism of cell death of an 8-aminoquinoline-naphthyl copper complex (Cu8AqN) in MCF-7 and MDA-MB-231 breast cancer cell lines was investigated. The complex inhibited the growth of MCF-7 and MDA-MB-231 cells with IC50 values of 2.54 ± 0.69 µM and 3.31 ± 0.06 µM, respectively. Nuclear fragmentation, annexin V binding, and increased caspase-3/7 activity indicated apoptotic cell death. The loss of mitochondrial membrane potential, an increase in caspase-9 activity, the absence of active caspase-8 and a decrease of tumour necrosis factor receptor 1(TNFR1) expression supported activation of the intrinsic apoptotic pathway. Increased ROS formation and increased expression of haem oxygenase-1 (HMOX-1) indicated activation of cellular stress pathways. Expression of p21 protein in the nuclei was increased indicating cell cycle arrest, whilst the expression of inhibitor of apoptosis proteins (IAPs); cIAP1, XIAP and survivin were decreased, creating a pro-apoptotic environment. Phosphorylated p53 species; phospho-p53(S15), phospho-p53(S46), and phospho-p53(S392) accumulated in MCF-7 cells indicating the potential of Cu8AqN to restore p53 function in the cells. In combination, the data indicates that Cu8AqN is a useful lead molecule worthy of further exploration as a potential anti-cancer drug.

Crystal structure and Hirshfeld surface of a penta-amine-copper(II) complex with urea and chloride.

The reaction of copper(II) oxalate and hexa-methyl-ene-tetra-mine in a deep eutectic solvent made of urea and choline chloride produced crystals of penta-amine-copper(II) dichloride-urea (1/1), [Cu(NH3)5]Cl2·CO(NH2)2, which was characterized by single-crystal X-ray diffraction. The complex contains discrete penta-amine-copper(II) units in a square-based pyramidal geometry. The overall structure of the multi-component crystal is dictated by hydrogen bonding between urea mol-ecules and amine H atoms with chloride anions.

Synthesis of Nano Sulfur/Chitosan-Copper Complex and Its Nematicidal Effect against Meloidogyne incognita In Vitro and on Coffee Pots.

Sulfur is one of the inorganic elements used by plants to develop and produce phytoalexin to resist certain diseases. This study reported a method for preparing a material for plant disease resistance. Sulfur nanoparticles (SNPs) stabilized in the chitosan-Cu2+ (CS-Cu2+) complex were synthesized by hydrolysis of Na2S2O3 in an acidic medium. The obtained SNPs/CS-Cu2+ complex consisting of 0.32% S, 4% CS, and 0.7% Cu (w/v), contained SNPs with an average size of ~28 nm as measured by transmission electron microscopy images. The X-ray diffraction pattern of the SNPs/CSCu2+ complex showed that SNPs had orthorhombic crystal structures. Interaction between SNPs and the CS-Cu2+ complex was also investigated by ultraviolet-visible. Results in vitro nematicidal effect of materials against Meloidogyne incognita showed that SNPs/CS-Cu2+ complex was more effective in killing second-stage juveniles (J2) nematodes and inhibiting egg hatching than that of CS and CS-Cu2+ complex. The values of LC50 in killing J2 nematodes and EC50 in inhibiting egg hatching of SNPs/CS-Cu2+ complex were 75 and 51 mg/l, respectively. These values were lower than those of CS and the CS-Cu2+ complex. The test results on the nematicidal effect against M. incognita on coffee pots showed that the SNPs/CS-Cu2+ complex was 100% effective at a concentration of 150 mg/l. Therefore, the SNPs/CS-Cu2+ complex could be considered as a biochemical material with potential for agricultural applications to control root-knot nematodes.

A Triply Linked Copper(III) Dicarbacorrole Dimer.

A triply linked dicarbacorrole dimer (7) was synthesized from a new meso-meso singly linked dicarbacorrole dimer precursor (6) via an oxidative fusion reaction by 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in the presence of trifluoromethanesulfonic acid (TfOH). Single crystal X-ray structure of 7 adopts a flat conformation with a length as ca. 15.946 Šand a width as 6.903 Å, which can be regarded as a short carbaporphyrinoid tape. Two coordinated Cu ions keeps the +3 oxidation state in 7, as confirmed by NMR spectroscopy, single crystal X-ray diffraction and X-ray photoelectron spectroscopy (XPS). This is in sharp contrast to the Osuka's triply linked tetrapyrrolic corrole dimers, where the inner 3NH form is not stable and thus can only act as a divalent ligand. Due to the non-aromatic nature of dicarbacorrole macrocycle, the largely decreased HOMO-LUMO gap and red-shifted absorption of 7 are best ascribed to the strong electronic interaction between two dipyrromethene-type chromophores. To our knowledge, this is the first fully fused carbaporphyrinoid dimer with ß-ß, meso-meso, ß-ß triply linkages prepared to date.

Secondary Structure in Enzyme-Inspired Polymer Catalysts Impacts Water Oxidation Efficiency.

Protein structure plays an essential role on their stability, functionality, and catalytic activity. In this work, the interplay between the ß-sheet structure and its catalytic implications to the design of enzyme-inspired materials is investigated. Here, inspiration is drawn from the active sites and ß-sheet rich structure of the highly efficient multicopper oxidase (MCO) to engineer a bio-inspired electrocatalyst for water oxidation utilizing the abundant metal, copper. Copper ions are coordinated to poly-histidine (polyCuHis), as they are in MCO active sites. The resultant polyCuHis material effectively promotes water oxidation with low overpotentials (0.15 V) in alkaline systems. This activity is due to the 3D structure of the poly-histidine backbone. By increasing the prevalence of ß-sheet structure and decreasing the random coil nature of the polyCuHis secondary structures, this study is able to modulates the electrocatalytic activity of this material is modulated, shifting it toward water oxidation. These results highlight the crucial role of the local environment at catalytic sites for efficient, energy-relevant transformations. Moreover, this work highlights the importance of conformational structure in the design of scaffolds for high-performance electrocatalysts.

Copper Nanodrugs by Atom Transfer Radical Polymerization.

In this study, some copper catalysts used for atom transfer radical polymerization (ATRP) were explored as efficient anti-tumor agents. The aqueous solution of copper-containing nanoparticles with uniform spheric morphology was in situ prepared through a copper-catalyzed activator generated by electron transfer (AGET) ATRP in water. Nanoparticles were then directly injected into tumor-bearing mice for antitumor chemotherapy. The copper nanodrugs had prolonged blood circulation time and enhanced accumulation at tumor sites, thus showing potent antitumor activity. This work provides a novel strategy for precise and large-scale preparation of copper nanodrugs with high antitumor activity.

Synthesis and Characterization of Slow-Release Chitosan Oligosaccharide Pyridine Schiff Base Copper Complexes with Antifungal Activity.

Herein, a series of chitosan oligosaccharide copper complexes modified with pyridine groups (CPSx-Cu complexes) were successfully prepared via the Schiff base reaction and ion complexation reaction for slow-release fungicide. The structures of the synthesized derivatives were characterized via Fourier transform infrared spectroscopy and 1H and 13C nuclear magnetic resonance spectroscopy, and the unit configuration of the complexes was calculated using Gaussian software. The slow-release performance experiment demonstrated that the cumulative copper ion release rate of CPSx-Cu complexes was dependent on the type of substituents on the pyridine ring. Furthermore, the in vitro and in vivo antifungal activities of the CPSx-Cu complexes were investigated. At a concentration of 0.4 mg/mL, CPSx-Cu complexes completely inhibited the growth of Pythium vexans and Phytophthora capsici. Results indicated that CPSx-Cu complexes with slow-release ability exhibited better antifungal activity than thiodiazole-copper and copper sulfate basic. This study confirmed that combining chitosan oligosaccharide with bioactive pyridine groups and copper ions is an effective approach to further developing slow-release copper fungicides, providing new possibilities for the application of copper fungicides in green agriculture. This study lays the foundation for further studies on biogreen copper fungicides.

Electrocatalytic Hydrogen Evolution of Immobilized Copper Complex on Carbonaceous Materials: From Neutral Water to Seawater.

Electrochemical hydrogen evolution reaction (HER) is an appealing strategy to utilize renewable electricity to produce green H2. Moreover, use of neutral-pH electrolyte such as water and seawater for the HER has long been desired for eco-friendly energy production that aligns with net zero emission goal. Herein, new heterogeneous catalysts were developed by dispersing an HER-active copper complex containing N4-Schiff base macrocycle (CuL) on carbonaceous materials, i. e. multi-walled carbon nanotube (CNT) and graphene oxide (GO), via non-covalent interaction and investigated their HER performance. It was found that CuL/GO exhibited higher HER activity than CuL/CNT, possibly due to its significantly larger amount of CuL immobilized onto GO. In addition, CuL/GO showed satisfactory HER performance in a neutral (pH 7) NaCl electrolyte solution. Notably, the performances of CuL/GO were boosted up when performed in natural seawater sample with the faradaic efficiency of 70 % and 3 times higher amount of H2 at -0.6 V vs reversible hydrogen electrode (RHE), in comparison to the HER in a NaCl electrolyte. Furthermore, it possessed a low overpotential of 139 mV at -10 mA/cm2. This demonstrated the potential use of CuL/GO as an effective HER catalyst in seawater for further sustainable development.

Copper complex molecules as dye-sensitizers: Hybrid MetaGGA and standard + van der Waals functionals.

This study focuses on the use of Density Functional Theory calculations with two main approaches: computational chemistry and computational physics. The following three cases were considered for the derivation: (I) computational chemistry using the M06 hybrid functional, (II) computational chemistry using the standard PBE functional including vdW interactions, and (III) computational physics using the standard PBE functional including vdW interactions and periodic boundary conditions. Since the approximation using hybrid functionals M06 has been extensively validated, this method was used as a reference. The second and third methods are less expensive, it is ideal for use to extend large systems. From the sensitized molecules are found in the gas phase and include solvent effects through the integral equation formalism polarizable continuum model. In a systematic analysis of 15 Cu complex molecules, a complete characterization for DSSCs has been carried out and molecular geometry, electronic and optical measurements have been reported.

Copper(II) complexes with plumbagin and bipyridines target mitochondria for enhanced chemodynamic cancer therapy.

The combination of mitochondrial targeting and chemodynamic therapy is a promising anti-cancer strategy. Three mitochondria targeting copper(II) complexes (Cu1-Cu3) with plumbagin and bipyridine ligands for enhanced chemodynamic therapy were synthesized and characterized. Their anti-proliferative activity to HeLa cells was higher than that of cisplatin, and their toxicity to normal cells was low. Cellular uptake and distribution studies indicated that Cu1 and Cu3 were mainly accumulated in mitochondria. The mechanism studies showed that Cu1 and Cu3 converted intracellular H2O2 into toxic hydroxyl radicals by consuming glutathione, leading to mitochondrial dysfunction. Treatment with the copper complex caused ER stress and cell arrest in the S phase which resulted in apoptosis. In vivo, Cu1 and Cu3 effectively inhibited the growth of HeLa xenograft tumors without obvious toxic and side effects.

Heterogenized Molecular Electrocatalyst Based on a Hydroxo-Bridged Binuclear Copper(II) Phenanthroline Compound for Selective Reduction of CO2 to Ethylene.

Molecular copper catalysts have emerged as promising candidates for the electrochemical reduction of CO2 . Notable features of such systems include the ability of Cu to generate C2+  products and the well-defined active sites that allow for targeted structural tuning. However, the frequently observed in situ formation of Cu nanoclusters has undermined the advantages of the molecular frameworks. It is therefore desirable to develop Cu-based catalysts that retain their molecular structures during electrolysis. In this context, a heterogenized binuclear hydroxo-bridged phenanthroline Cu(II) compound with a short Cu···Cu distance is reported as a simple yet efficient catalyst for electrogeneration of ethylene and other C2 products. In an aqueous electrolyte, the catalyst demonstrates remarkable performance, with excellent Faradaic efficiency for C2 products (62%) and minimal H2 evolution (8%). Furthermore, it exhibits high stability, manifested by no observable degradation during 15 h of continuous electrolysis. The preservation of the atomic distribution of the active sites throughout electrolysis is substantiated through comprehensive characterizations, including X-ray photoelectron and absorption spectroscopy, scanning and transmission electron microscopy, UV-vis spectroscopy, as well as control experiments. These findings establish a solid foundation for further investigations into targeted structural tuning, opening new avenues for enhancing the catalytic performance of Cu-based molecular electrocatalysts.

Magnetically-assisted electrochemical immunoplatform for simultaneous detection of active and total prostate-specific antigen based on proteolytic reaction and sandwich affinity analysis.

Simultaneous detection of active and inactive proteases is clinically meaningful for improving diagnostic specificity. In this work, we reported an electrochemical method for simultaneous immunoassays of active and total proteases. Magnetic beads (MBs) were used as the solid supports for immobilization of capture antibodies and enrichment of targets. For the detection of active protease, the proteolytic-reaction-based analysis was carried out by the generation of Cu2+-binding peptide, in which a label-free peptide was used as the proteolytic substrate. The redox potential of the resulting peptide-Cu2+ complex was intrinsically distinguished from that of free Cu2+, thus allowing the "signal-on" detection of active protease. For the immunoassay of total protease in a sandwich-like format, electroactive metal-organic frameworks (Cu-MOFs) were used as the signal tags. The captured Cu-MOFs could directly produce a well-defined electrochemical signal from the reduction of Cu2+ ions. The analytical performances of the immunoplatform were evaluated by determining the model analytes of free and total prostate-specific antigen (fPSA and tPSA) in buffer and serum. The detection limits were found to be 0.3 pM for fPSA and 2 pM for tPSA. This work proposed a new strategy for simultaneous detection of active and total proteases, which should be evaluable for clinical diagnosis and treatment of protease-relative diseases.