Multiresponsive Core-Shell Microgels Functionalized by Nitrilotriacetic Acid.

Stimuli-responsive microgels with ionizable functional groups offer versatile applications, e.g., by the uptake of oppositely charged metal ions or guest molecules such as drugs, dyes, or proteins. Furthermore, the incorporation of carboxylic groups enhances mucoadhesive properties, crucial for various drug delivery applications. In this work, we successfully synthesized poly{N-vinylcaprolactam-2,2'-[(5-acrylamido-1-carboxypentyl)azanediyl]diacetic acid} [p(VCL/NTAaa)] microgels containing varying amounts of nitrilotriacetic acid (NTA) using precipitation polymerization. We performed fundamental characterization by infrared (IR) spectroscopy and dynamic and electrophoretic light scattering. Despite their potential multiresponsiveness, prior studies on NTA-functionalized microgels lack in-depth analysis of their stimuli-responsive behavior. This work addresses this gap by assessing the microgel responsiveness to temperature, ionic strength, and pH. Morphological investigations were performed via NMR relaxometry, nanoscale imaging (AFM and SEM), and reaction calorimetry. Finally, we explored the potential application of the microgels by conducting cytocompatibility experiments and demonstrating the immobilization of the model protein cytochrome c in the microgels.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Exploring the Antitumor Efficacy of N-Heterocyclic Nitrilotriacetate Oxidovanadium(IV) Salts on Prostate and Breast Cancer Cells.

The crystal structures of two newly synthesized nitrilotriacetate oxidovanadium(IV) salts, namely [QH][VO(nta)(H2O)](H2O)2 (I) and [(acr)H][VO(nta)(H2O)](H2O)2 (II), were determined. Additionally, the cytotoxic effects of four N-heterocyclic nitrilotriacetate oxidovanadium(IV) salts-1,10-phenanthrolinium, [(phen)H][VO(nta)(H2O)](H2O)0.5 (III), 2,2'-bipyridinium [(bpy)H][VO(nta)(H2O)](H2O) (IV), and two newly synthesized compounds (I) and (II)-were evaluated against prostate cancer (PC3) and breast cancer (MCF-7) cells. All the compounds exhibited strong cytotoxic effects on cancer cells and normal cells (HaCaT human keratinocytes). The structure-activity relationship analysis revealed that the number and arrangement of conjugated aromatic rings in the counterion had an impact on the antitumor effect. The compound (III), the 1,10-phenanthrolinium analogue, exhibited the greatest activity, whereas the acridinium salt (II), with a different arrangement of three conjugated aromatic rings, showed the lowest toxicity. The increased concentrations of the compounds resulted in alterations to the cell cycle distribution with different effects in MCF-7 and PC3 cells. In MCF-7 cells, compounds I and II were observed to block the G2/M phase, while compounds III and IV were found to arrest the cell cycle in the G0/G1 phase. In PC3 cells, all compounds increased the rates of cells in the G0/G1 phase.

Development of a Modular NTA:His Tag Viral Vaccine for Co-delivery of Antigen and Adjuvant.

The SARS-CoV-2 pandemic has highlighted the need for vaccines that are effective, but quickly produced. Of note, vaccines with plug-and-play capabilities that co-deliver antigen and adjuvant to the same cell have shown remarkable success. Our approach of utilizing a nitrilotriacetic acid (NTA) histidine (His)-tag chemistry with viral adjuvants incorporates both of these characteristics: plug-and-play and co-delivery. We specifically utilize the cowpea mosaic virus (CPMV) and the virus-like particles from bacteriophage Qß as adjuvants and bind the model antigen ovalbumin (OVA). Successful binding of the antigen to the adjuvant/carrier was verified by SDS-PAGE, western blot, and ELISA. Immunization in C57BL/6J mice demonstrates that with Qß - but not CPMV - there is an improved antibody response against the target antigen using the Qß-NiNTA:His-OVA versus a simple admixture of antigen and adjuvant. Antibody isotyping also shows that formulation of the vaccines can alter T helper biases; while the Qß-NiNTA:His-OVA particle produces a balanced Th1/Th2 bias the admixture was strongly Th2. In a mouse model of B16F10-OVA, we further demonstrate improved survival and slower tumor growth in the vaccine groups compared to controls. The NiNTA:His chemistry demonstrates potential for rapid development of future generation vaccines enabling plug-and-play capabilities with effectiveness boosted by co-delivery to the same cell.

A Simple and Versatile Strategy for Oriented Immobilization of His-Tagged Proteins on Magnetic Nanoparticles.

Oriented and covalent immobilization of proteins on magnetic nanoparticles (MNPs) is particularly challenging as it requires both the functionality of the protein and the colloidal stability of the MNPs to be preserved. Here, we describe a simple, straightforward, and efficient strategy for MNP functionalization with proteins using metal affinity binding. Our method involves a single-step process where MNPs are functionalized using a preformed, ready-to-use nitrilotriacetic acid-divalent metal cation (NTA-M2+) complex and polyethylene glycol (PEG) molecules. As a proof-of-concept, we demonstrate the oriented immobilization of a recombinant cadherin fragment engineered with a hexahistidine tag (6His-tag) onto the MNPs. Our developed methodology is simple and direct, enabling the oriented bioconjugation of His-tagged cadherins to MNPs while preserving protein functionality and the colloidal stability of the MNPs, and could be extended to other proteins expressing a polyhistidine tag. When compared to the traditional method where NTA is first conjugated to the MNPs and afterward free metal ions are added to form the complex, this novel strategy results in a higher functionalization efficiency while avoiding MNP aggregation. Additionally, our method allows for covalent bonding of the cadherin fragments to the MNP surface while preserving functionality, making it highly versatile. Finally, our strategy not only ensures the correct orientation of the protein fragments on the MNPs but also allows for the precise control of their density. This feature enables the selective targeting of E-cadherin-expressing cells only when MNPs are decorated with a high density of cadherin fragments.

Mapping the electronic transitions of protonation sites in peptides using soft X-ray action spectroscopy.

Near-edge X-ray absorption mass spectrometry (NEXAMS) around the nitrogen and oxygen K-edges was employed on gas-phase peptides to probe the electronic transitions related to their protonation sites, namely at basic side chains, the N-terminus and the amide oxygen. The experimental results are supported by replica exchange molecular dynamics and density-functional theory and restricted open-shell configuration with single calculations to attribute the transitions responsible for the experimentally observed resonances. We studied five tailor-made glycine-based pentapeptides, where we identified the signature of the protonation site of N-terminal proline, histidine, lysine and arginine, at 406 eV, corresponding to N 1s → σ*(NHx+) (x = 2 or 3) transitions, depending on the peptides. We compared the spectra of pentaglycine and triglycine to evaluate the sensitivity of NEXAMS to protomers. Separate resonances have been identified to distinguish two protomers in triglycine, the protonation site at the N-terminus at 406 eV and the protonation site at the amide oxygen characterized by a transition at 403.1 eV.

Comparison of synthetic and organic biodegradable chelants in augmenting cadmium phytoextraction in Solanum nigrum.

This study focused to enhance the cadmium (Cd) phytoextraction efficiency in Solanum nigrum by applying four biodegradable chelants (10 mM)-ethylene glycol tetraacetic acid (EGTA), ethylenediamine disuccinate (EDDS), nitrilotriacetic acid (NTA), and citric acid (CA), when grown in Cd-spiked soil (12 and 48 mg kg-1). Plant height, dry biomass, photosynthetic traits, and metal accumulation varied significantly with Cd and chelant treatments. Cadmium-toxicity resulted in reduction of plant growth and photosynthetic physiology, whereas chelant supplementation alleviated the toxic effect of Cd and increased its accumulation. Tolerance index value increased with addition of chelants in the order: EGTA (1.57-1.63) >EDDS (1.39-1.58) >NTA (1.14-1.50) >CA (1-1.22) compared with Cd (0.46-1.08). Transfer coefficient of root increased with supplementation of EGTA (3.40-3.85), EDDS (3.10-3.40), NTA (2.60-2.90), and CA (1.85-2.29), over Cd-alone (1.61-1.63). Similarly, translocation factor was also increased upon addition of EGTA (0.52-0.73), EDDS (0.35-0.81), NTA (0.38-0.75), and CA (0.53-0.54), compared with Cd-alone (0.36-0.59). Maximum Cd removal (67.67% at Cd12 and 36.05% at Cd48) was observed with supplementation of EGTA. The study concludes that the supplementation of EGTA and EDDS with S. nigrum can be employed as an efficient and environmentally safe technique for reclamation of Cd-contaminated soils.

Apart from the selection of a good hyperaccumulator, the choice of chelant (biodegradable/non-biodegradable) is an important aspect for the successful phytoextraction of metals from contaminated soil. We reported for the first time the potential of ethylene glycol tetraacetic acid (EGTA; a biodegradable chelant) in enhancing Cd phytoextraction by Solanum nigrum. Comparative appraisal of metal extraction efficiency of biodegradable chelants at low (12 mg kg⁻¹) and high (48 mg kg⁻¹) Cd dose depicted that EGTA performed better than EDDS, NTA, and CA (other biodegradable chelants). EGTA supplementation did not induce toxicity in plants; rather it improved metal accumulation, morphology, and photosynthetic physiology.

Strategies for Mitigating Commercial Sensor Chip Variability with Experimental Design Controls.

Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.

Switch-on Fluorescence Analysis of Protease Activity with the Assistance of a Nickel Ion-Nitrilotriacetic Acid-Conjugated Magnetic Nanoparticle.

Heterogeneous protease biosensors show high sensitivity and selectivity but usually require the immobilization of peptide substrates on a solid interface. Such methods exhibit the disadvantages of complex immobilization steps and low enzymatic efficiency induced by steric hindrance. In this work, we proposed an immobilization-free strategy for protease detection with high simplicity, sensitivity and selectivity. Specifically, a single-labeled peptide with oligohistidine-tag (His-tag) was designed as the protease substrate, which can be captured by a nickel ion-nitrilotriacetic acid (Ni-NTA)-conjugated magnetic nanoparticle (MNP) through the coordination interaction between His-tag and Ni-NTA. When the peptide was digested by protease in a homogeneous solution, the signal-labeled segment was released from the substrate. The unreacted peptide substrates could be removed by Ni-NTA-MNP, and the released segments remained in solution to emit strong fluorescence. The method was used to determine protease of caspase-3 with a low detection limit (4 pg/mL). By changing the peptide sequence and signal reporters, the proposal could be used to develop novel homogeneous biosensors for the detection of other proteases.

Comparative assessment of two biodegradable chelants, S,S-ethylenediamine disuccinic acid and nitrilotriacetic acid, in facilitating Cd remediation by lesser swine cress (Coronopus didymus, Brassicaceae).

Chemically assisted phytoremediation is suggested as an effective approach to amplify the metal-remediating potential of hyperaccumulators. The current study assessed the efficiency of two biodegradable chelants (S,S-ethylenediamine disuccinic acid, EDDS; nitrilotriacetic acid, NTA) in enhancing the remediation of Cd by Coronopus didymus (Brassicaceae). C. didymus growing in Cd-contaminated soil (35-175 mg kg-1 soil) showed increased growth and biomass due to the hormesis effect, and chelant supplementation further increased growth, biomass, and Cd accumulation. A significant interaction with chelants and different Cd concentrations was observed, except for Cd content in roots and Cd content in leaves, which exhibited a non-significant interaction with chelant addition. The effect of the NTA amendment on the root dry biomass and shoot dry biomass was more pronounced than EDDS at all the Cd treatments. Upon addition of EDDS and NTA, bio-concentration factor values were enhanced by ~184-205 and ~ 199-208, respectively. The tolerance index of root and shoot increased over the control upon the addition of chelants, with NTA being better than EDDS. With chelant supplementation, bio-accumulation coefficient values were in the order Cd35 + NTA (~163%) > Cd105 + NTA (~137%) > Cd35 + EDDS (~89%) > Cd175 + NTA (~85%) > Cd105 + EDDS (~62%) > Cd175 + EDDS (~40%). The translocation factor correlated positively (r ≥ 0.8) with tolerance index and Cd accumulation in different plant parts. The study demonstrated that chelant supplementation enhanced Cd-remediation efficiency in C. didymus as depicted by improved plant growth and metal accumulation, and NTA was more effective than EDDS in reclaiming Cd.

Ferroptosis resistance determines high susceptibility of murine A/J strain to iron-induced renal carcinogenesis.

Cancer susceptibility is a critical factor in the understanding of carcinogenesis. Intraperitoneal (i.p.) injection of an iron chelate, ferric nitrilotriacetate (Fe-NTA), produces hydroxyl radicals via Fenton reaction to induce ferroptosis in renal proximal tubules. Rats or mice subjected to repeated i.p. injections of Fe-NTA develop renal cell carcinoma (RCC). To elucidate the molecular mechanisms that cause susceptibility to renal carcinogenesis, we first established an inter-strain difference in the susceptibility to Fe-NTA-induced renal carcinogenesis in mice. Based on a previous observation of a low incidence of RCC with this model in C57BL/6J strain mice, we investigated A/J strain mice here, which demonstrated significantly higher susceptibility to Fe-NTA-induced renal carcinogenesis. Homozygous deletion of the Cdkn2a/2b tumor suppressor locus was detected for the first time in A/J strain mice. Focusing on ferroptosis and iron metabolism, we explored the mechanisms involved that lead to the difference in RCC development. We compared the protective responses in the kidney of A/J and C57BL/6J strains after Fe-NTA treatment. After 3-week Fe-NTA treatment, A/J mice maintained higher levels of expression of glutathione peroxidase 4 and xCT (SLC7A11), leading to a lower level of lipid peroxidation. Simultaneously, A/J mice had decreased expression of transferrin receptor and increased expression of ferritin to greater degrees than C57BL/6 mice. After a single Fe-NTA injection, higher levels of oxidative cell damage and cytosolic catalytic Fe(II) were observed in C57BL/6J mice, accompanied by a greater increase in lipocalin-2. Lipocalin-2 deficiency significantly decreased oxidative renal damage. Our results suggest that a genetic trait favoring ferroptosis resistance contributes to high susceptibility to Fe-NTA-induced RCC in A/J strain.

Tyrosine-Based Dual-Functional Interface for Trapping and On-Site Photo-Induced Covalent Immobilization of Proteins.

Tyrosine, a simple and well-available natural amino acid, is featured by the small size of the compound that contains multiple reactive groups. This study developed an efficient bioconjugation strategy using tyrosine-based dual-functional interfaces. When tyrosine molecules are immobilized on the surface of a supporting material through amino groups, their carboxyl groups can function as an attracting trap due to their anionic nature at neutral pH and ability to chelate nickel(II) ions (Ni2+), allowing the capture and enrichment of cationic proteins and histidine (His)-tagged proteins on the surface. The trapped proteins can be further covalently immobilized on site through ruthenium-mediated photochemical cross-linking, which has been found to be highly efficient and can be completed within minutes. This strategy was successfully applied to two different material systems. We found that tyrosine-modified agarose beads had a binding capacity of the His-tagged enhanced green fluorescent protein comparable to that of commonly used nitrilotriacetic acid-based resins, and further covalent coupling via dityrosine cross-linking achieved a yield of 85% within 5 min, without compromising much on its fluorescence activity. On the surface of tyrosine-modified 316L stainless steel, lysozyme was captured through electrostatic interaction and further immobilized. The resultant surface exhibited remarkable antibacterial activity against both Staphylococcus aureus and Escherichia coli. Such a tyrosine-based capture-then-coupling method is featured by its simplicity, high coupling efficiency, and high utilization rate of target molecules, making it particularly suitable for the proteins that are highly priced or vulnerable to general immobilization chemistry.

Artificial Nanocage Formed via Self-Assembly of ß-Annulus Peptide for Delivering Biofunctional Proteins into Cell Interiors.

Nanocarriers that deliver functional proteins to cell interiors are an attractive platform for the intracellular delivery of intact proteins without further modification, with in vivo compatibility. Development of efficient methods for cargo protein encapsulation and release in recipient cell cytosol is needed. Herein, we assess the feasibility of the abovementioned requirements using a protein nanocage (artificial nanocage) without compromising the structure and functions of the original protein and allowing for design flexibility of the surfaces and interiors. The protein nanocage formed via the self-assembly of the ß-annulus peptide (24-amino acid peptide) in water was used as a model framework. The nitrilotriacetic acid moiety was displayed on the nanocage lumen for effective encapsulation of hexahistidine-tagged proteins in the presence of Ni2+, and the amphiphilic cationic lytic peptide HAad was displayed on a nanocage surface to attain cell permeability. Successful intracellular delivery of cargo proteins and targeting of cytosolic proteins by a nanobody were achieved, indicating the validity of the approach employed in this study.

The impact of the electric field of metal ions on the vibrations and internal hydrogen bond strength in alkali metal ion di- and triglycine complexes.

Using infrared predissociation spectroscopy of cryogenic ions, we revisit the vibrational spectra of alkali metal ion (Li+, Na+, K+) di- and triglycine complexes. We assign their most stable conformation, which involves metal ion coordination to all C=O groups and an internal NH⋯NH2 hydrogen bond in the peptide backbone. An analysis of the spectral shifts of the OH and C=O stretching vibrations across the different metal ions and peptide chain lengths shows that these are largely caused by the electric field of the metal ion, which varies in strength as a function of the square of the distance. The metal ion-peptide interaction also remotely modulates the strength of internal hydrogen bonding in the peptide backbone via the weakening of the amide C=O bond, resulting in a decrease in internal hydrogen bond strength from Li+ > Na+ > K+.

Encapsulation of hydrophobic components in dendrimersomes and decoration of their surface with proteins and nucleic acids.

Reconstructing the functions of living cells using nonnatural components is one of the great challenges of natural sciences. Compartmentalization, encapsulation, and surface decoration of globular assemblies, known as vesicles, represent key early steps in the reconstitution of synthetic cells. Here we report that vesicles self-assembled from amphiphilic Janus dendrimers, called dendrimersomes, encapsulate high concentrations of hydrophobic components and do so more efficiently than commercially available stealth liposomes assembled from phospholipid components. Multilayer onion-like dendrimersomes demonstrate a particularly high capacity for loading low-molecular weight compounds and even folded proteins. Coassembly of amphiphilic Janus dendrimers with metal-chelating ligands conjugated to amphiphilic Janus dendrimers generates dendrimersomes that selectively display folded proteins on their periphery in an oriented manner. A modular strategy for tethering nucleic acids to the surface of dendrimersomes is also demonstrated. These findings augment the functional capabilities of dendrimersomes to serve as versatile biological membrane mimics.

Efficiency of heterogeneous chelating agents on the phytoremediation potential and growth of Sasa argenteostriata (Regel) E.G. Camus on Pb-contaminated soil.

Ethylenediaminetetraacetic acid (EDTA) is one of the most effective chelating agents for enhancing lead (Pb) accumulation in various plant organs. However, it has a higher risk of causing secondary pollution than other chelating agents. To reduce such environmental risks and increase remediation efficiency, EDTA can be combined with degradable chelating agents for use in phytoremediation, but there are few reports on the combination of EDTA and nitrilotriacetic acid (NTA). This study evaluated the effects of combined EDTA and NTA application at different concentrations (900, 1200, or 1500 mg/kg) and with different methods (1 application or 3 applications) on dwarf bamboo (Sasa argenteostriata (Regel) E.G. Camus) growth and phytoremediation efficiency and on the soil environment in pot experiments with Pb-contaminated soil. Applying EDTA and NTA together resulted in lower soil water-soluble Pb concentrations than applying EDTA alone and therefore resulted in lower environmental risk. The increased availability of soil Pb produced a stress response in the dwarf bamboo plants, which increased their biomass significantly. Moreover, under the chelating treatments, the soil Pb availability increased, which promoted Pb translocation in plants. The Pb content in the aerial parts of the dwarf bamboo increased significantly in all treatments (translocation factors increased by 300~1500% compared with that in CK). The Pb content increase in the aerial parts caused high proline accumulation in dwarf bamboo leaves, to alleviate Pb toxicity. Maximum Pb accumulation was observed in the EN1500 treatment, which was significantly higher than that in the other treatments except the EN900 treatment. This study elucidates the choice of remediation techniques and the physiological characteristics of the plants used in such studies. In conclusion, the EN900 treatment resulted in the lowest environmental risk, greatest biomass production, and highest phytoremediation efficiency of all treatments, indicating that it has great potential for application in phytoremediation with dwarf bamboo in Pb-contaminated soil.

Effect of intra-nasal nitrilotriacetic acid trisodium salt in lowering elevated calcium cations and improving olfactory dysfunction in COVID-19 patients.

PURPOSE: An association between COVID-19 and olfactory dysfunction has been noted in many patients worldwide. The olfactory adaptation process leads to an increase in intracellular calcium cation levels. Nitrilotriacetic acid trisodium salt has high selective chelation for calcium cations from olfactory mucus. The aim of this work is to test the effect of an intranasal nitrilotriacetic acid trisodium salt to lower the elevated calcium cations in COVID-19 patients with relevant symptoms of olfactory dysfunction. METHODS: Fifty-eight COVID-19 adult patients with relevant symptoms of olfactory dysfunction were enrolled in a prospective randomized controlled trial. They received a nasal spray containing either 0.9% sodium chloride or 2% nitrilotriacetic acid trisodium salt. Olfactory function was assessed before and after treatment using the Sniffin' Sticks test. Quantitative analysis of calcium cation concentration in nasal secretions was performed using a carbon paste ion-selective electrode. RESULTS: After the application of nitrilotriacetic acid trisodium salt compared to sodium chloride, a significant improvement from functional anosmia to healthy normosmia with significant decrease in calcium cation concentration was observed. CONCLUSIONS: Further collaborative research is needed to fully investigate the effect of an intranasal nitrilotriacetic acid trisodium salt in the treatment of olfactory disorders.

The Interplay of Conjugation and Metal Coordination in Tuning the Electron Transfer Abilities of NTA-Graphene Based Interfaces.

An artificial leaf is a concept that not only replicates the processes taking place during natural photosynthesis but also provides a source of clean, renewable energy. One important part of such a device are molecules that stabilize the connection between the bioactive side and the electrode, as well as tune the electron transfer between them. In particular, nitrilotriacetic acid (NTA) derivatives used to form a self-assembly monolayer chemisorbed on a graphene monolayer can be seen as a prototypical interface that can be tuned to optimize the electron transfer. In the following work, interfaces with modifications of the metal nature, backbone saturation, and surface coverage density are presented by means of theoretical calculations. Effects of the type of the metal and the surface coverage density on the electronic properties are found to be key to tuning the electron transfer, while only a minor influence of backbone saturation is present. For all of the studied interfaces, the charge transfer flow goes from graphene to the SAM. We suggest that, in light of the strength of electron transfer, Co2+ should be considered as the preferred metal center for efficient charge transfer.

An antibody-free dual-biomarker rapid enrichment workflow (AnDREW) improves the sensitivity of malaria rapid diagnostic tests.

Rapid diagnostic tests (RDTs) are critical to the success of malaria elimination campaigns. These tests are rapid, user-friendly, and field-deployable to resource-limited regions. However, RDTs demonstrate poor sensitivity because they can only tolerate a small (5 µL) volume of blood, which limits the amount of protein biomarker delivered to the test. We have developed the Antibody-free Dual-biomarker Rapid Enrichment Workflow (AnDREW) for purifying histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (PLDH) from large volume (150 µL) blood samples. We used Zn(II)NTA and aptamer-conjugated magnetic beads to capture HRP2 and PLDH, respectively. Both biomarkers were then eluted into RDT-compatible volumes using ethylene diamine tetraacetic acid (EDTA). We optimized both bead conjugates individually by enzyme-linked immunosorbent assays (ELISAs) and then combined the optimized capture and elution assays for both biomarkers to produce the AnDREW. The AnDREW-enhanced RDTs exhibited a 11-fold and 9-fold improvement in analytical sensitivity for detection of HRP2 and PLDH, respectively, when compared to unenhanced RDTs. Moreover, the limit of detection for PLDH was improved 11-fold for the AnDREW-enhanced RDTs (3.80 parasites/µL) compared to unenhanced RDTs (42.31 parasites/µL). Importantly, the AnDREW utilizes a pan-specific PLDH aptamer and improves upon existing methods by eluting both biomarkers without complexed antibodies.

Site-specific covalent labeling of His-tag fused proteins with N-acyl-N-alkyl sulfonamide reagent.

The ability to incorporate a desired functionality into proteins of interest in a site-specific manner can provide powerful tools for investigating biological systems and creating therapeutic conjugates. However, there are not any universal methods that can be applied to all proteins, and it is thus important to explore the chemical strategy for protein modification. In this paper, we developed a new reactive peptide tag/probe pair system for site-specific covalent protein labeling. This method relies on the recognition-driven reaction of a peptide tag and a molecular probe, which comprises the lysine-containing short histidine tag (KH6 or H6K) and a binuclear nickel (II)- nitrilotriacetic acid (Ni2+-NTA) complex probe containing a lysine-reactive N-acyl-N-alkyl sulfonamide (NASA) group. The selective interaction of the His-tag and Ni2+-NTA propeles a rapid nucleophilic reaction between a lysine residue of the tag and the electrophilic NASA group of the probe by the proximity effect, resulting in the tag-site-specific functionalization of proteins. We characterized the reactive profile and site-specificity of this method using model peptides and proteins in vitro, and demonstrated the general utility for production of a nanobody-chemical probe conjugate without compromising its binding ability.