Iron Oxide Nanoparticle Clusters Research Articles

Shape and aspect ratio analysis of anisotropic magnetic nanochains based on TEM micrographs

Due to advances in electron microscopy and to the development of novel nanoparticle structures with different morphologies and the dependence of physical properties on the nanoparticle morphology, there is a need for a more precise analysis of nanoparticle structure and morphology. That should provide a simple and unambiguous comparison of nanoparticles‘ shapes and of material properties that depend on the shape, which has been lacking thus far. Here we study nanochains consisting of silica-coated iron oxide (maghemite, γ-Fe2O3) nanoparticle clusters covered by an additional layer of silica (core-shell structure). We have developed an algorithm for image segmentation and a quantitative analysis of nanochain shape from real TEM images. To that end we used two distinct measures of circularity and elongation measure (the aspect ratio measure). We show that the relative position and the area of the links, as well as the links‘ shape lead to significant differences in the measured aspect ratio of the entire nanochain (substantially influence the elongation of nanochains). We have also analyzed the core-shell structures in nanochains, and computed the shell's share in the overall area of observed nanochains. A Matlab code was developed and used for the computation of the elongation measure of shapes appearing in electron microscopy images. Here we have investigated magnetic properties of synthetic nanochains, that revealed superparamagnetic behavior at room temperature (SPION) with the possibility of tuning the magnetization values (approx. from 19 to 46 emu/g). We have compared of magnetization M(H) curves of the anisotropic nanochains and of isotropic nanoparticle (nanochain links), with the conclusion that the nanochains have a higher magnetic susceptibility, which fact can be understood as a consequence of their anisotropic shapes. The nanochains may be applied in biomedicine and magnetic separation, due to their morphology and magnetic properties.

Acetate-Induced Disassembly of Spherical Iron Oxide Nanoparticle Clusters into Monodispersed Core-Shell Structures upon Nanoemulsion Fusion.

It has been long known that the physical encapsulation of oleic acid-capped iron oxide nanoparticles (OA–IONPs) with the cetyltrimethylammonium (CTA+) surfactant induces the formation of spherical iron oxide nanoparticle clusters (IONPCs). However, the behavior and functional properties of IONPCs in chemical reactions have been largely neglected and are still not well-understood. Herein, we report an unconventional ligand-exchange function of IONPCs activated when dispersed in an ethyl acetate/acetate buffer system. The ligand exchange can successfully transform hydrophobic OA–IONP building blocks of IONPCs into highly hydrophilic, acetate-capped iron oxide nanoparticles (Ac–IONPs). More importantly, we demonstrate that the addition of silica precursors (tetraethyl orthosilicate and 3-aminopropyltriethoxysilane) to the acetate/oleate ligand-exchange reaction of the IONPs induces the disassembly of the IONPCs into monodispersed iron oxide–acetate–silica core–shell–shell (IONPs@acetate@SiO2) nanoparticles. Our observations evidence that the formation of IONPs@acetate@SiO2 nanoparticles is initiated by a unique micellar fusion mechanism between the Pickering-type emulsions of IONPCs and nanoemulsions of silica precursors formed under ethyl acetate buffered conditions. A dynamic rearrangement of the CTA+–oleate bilayer on the IONPC surfaces is proposed to be responsible for the templating process of the silica shells around the individual IONPs. In comparison to previously reported methods in the literature, our work provides a much more detailed experimental evidence of the silica-coating mechanism in a nanoemulsion system. Overall, ethyl acetate is proven to be a very efficient agent for an effortless preparation of monodispersed IONPs@acetate@SiO2 and hydrophilic Ac–IONPs from IONPCs.

Interaction Effects in Assembly of Magnetic Nanoparticles

A specific absorption rate of a dilute assembly of various random clusters of iron oxide nanoparticles in alternating magnetic field has been calculated using Landau–Lifshitz stochastic equation. This approach simultaneously takes into account both the presence of thermal fluctuations of the nanoparticle magnetic moments and magneto-dipole interaction between the nanoparticles of the clusters. It is shown that for usual 3D clusters, the intensity of the magneto-dipole interaction is determined mainly by the cluster packing density η = NpV/Vcl, where Np is the average number of the particles in the cluster, V is the nanoparticle volume, and Vcl is the cluster volume. The area of the low frequency hysteresis loop and the assembly-specific absorption rate have been found to be considerably reduced when the packing density of the clusters increases in the range of 0.005 ≤ η < 0.4. The dependence of the specific absorption rate on the mean nanoparticle diameter is retained with an increase of η, but becomes less pronounced. For fractal clusters of nanoparticles, which arise in biological media, in addition to a considerable reduction of the absorption rate, the absorption maximum is shifted to smaller particle diameters. It is found also that the specific absorption rate of fractal clusters increases appreciably with an increase of the thickness of nonmagnetic shells at the nanoparticle surfaces.

Photoacoustic-Guided Surgery with Indocyanine Green-Coated Superparamagnetic Iron Oxide Nanoparticle Clusters.

A common cause of local tumor recurrence in brain tumor surgery results from incomplete surgical resection. Adjunctive technologies meant to facilitate gross total resection have had limited efficacy to date. Contrast agents used to delineate tumors preoperatively cannot be easily or accurately used in the real-time operative setting. Although multimodal imaging contrast agents are developed to help the surgeon discern tumor from normal tissue in the operating room, these contrast agents are not readily translatable. This study has developed a novel contrast agent comprised solely of two Food and Drug Administration approved components, indocyanine green (ICG) and superparamagnetic iron oxide (SPIO) nanoparticles-with no additional amphiphiles or carrier materials, to enable preoperative detection by magnetic resonance (MR) imaging and intraoperative photoacoustic (PA) imaging. The encapsulation efficiency of both ICG and SPIO within the formulated clusters is ≈100%, and the total ICG payload is 20-30% of the total weight (ICG + SPIO). The ICG-SPIO clusters are stable in physiologic conditions; can be taken up within tumors by enhanced permeability and retention; and are detectable by MR. In a preclinical surgical resection model in mice, following injection of ICG-SPIO clusters, animals undergoing PA-guided surgery demonstrate increased progression-free survival compared to animals undergoing microscopic surgery.

Simultaneous surface and bulk sensitive XAS measurements of magnetic particle clusters

Magnetic iron oxide nanoparticle clusters (mnpc) coated with organic stabilizers were investigated using scanning transmission x-ray microscopy (STXM). Simultaneous surface and bulk sensitive Fe L3 edge absorption spectra, obtained using a photomultiplier tube and a channeltron, were used to detect subtle changes in the oxidation state in the surface and bulk of Iron Oxide mnpc. The effectiveness of this mode of STXM operation is demonstrated for these nanoparticle clusters.

Retention of ferrofluid aggregates at the target site during magnetic drug targeting

The present study reports the retention dynamics of a ferrofluid aggregate localized at the target site inside a glass capillary (500×500µm2 square cross section) against a bulk flow of DI water (Re=0.16 and 0.016) during the process of magnetic drug targeting (MDT). The dispersion dynamics of iron oxide nanoparticles (IONPs) into bulk flow for different initial size of aggregate at the target site is reported using the brightfield visualization technique. The flow field around the aggregate during the retention is evaluated using the µPIV technique. IONPs at the outer boundary experience a higher shear force as compared to the magnetic force, resulting in dispersion of IONPs into the bulk flow downstream to the aggregate. The blockage effect and the roughness of the outer boundary of the aggregate resulting from chain like clustering of IONPs contribute to the flow recirculation at the downstream region of the aggregate. The entrapment of seeding particles inside the chain like clusters of IONPs at the outer boundary of the aggregate reduces the degree of roughness resulting in a streamlined aggregate at the target site at later time. The effect of blockage, structure of the aggregate, and disturbed flow such as recirculation around the aggregate are the primary factors, which must be investigated for the effectiveness of the MDT process for in vivo applications.

Individually Stabilized, Superparamagnetic Nanoparticles with Controlled Shell and Size Leading to Exceptional Stealth Properties and High Relaxivities.

Superparamagnetic iron oxide nanoparticles (SPION) have received immense interest for biomedical applications, with the first clinical application as negative contrast agent in magnetic resonance imaging (MRI). However, the first generation MRI contrast agents with dextran-enwrapped, polydisperse iron oxide nanoparticle clusters are limited to imaging of the liver and spleen; this is related to their poor colloidal stability in biological media and inability to evade clearance by the reticuloendothelial system. We investigate the qualitatively different performance of a new generation of individually PEG-grafted core–shell SPION in terms of relaxivity and cell uptake and compare them to benchmark iron oxide contrast agents. These PEG-grafted SPION uniquely enable relaxivity measurements in aqueous suspension without aggregation even at 9.4 T magnetic fields due to their extraordinary colloidal stability. This allows for determination of the size-dependent scaling of relaxivity, which is shown to follow a d2 dependence for identical core–shell structures. The here introduced core–shell SPION with ∼15 nm core diameter yield a higher R2 relaxivity than previous clinically used contrast agents as well as previous generations of individually stabilized SPION. The colloidal stability extends to control over evasion of macrophage clearance and stimulated uptake by SPION functionalized with protein ligands, which is a key requirement for targeted MRI.

The effect of neighbor distance of magnetic nanoparticle clusters on magnetic resonance relaxation properties

Superparamagnetic iron oxide (SPIO) nanoparticle clusters are one unique form which can enhance magnetic relaxivity and improve the magnetic resonance imaging contrast at the same iron concentration, comparing to single SPIO nanoparticles. Controlling of cluster size and other structural parameters have drawn great interests in this field to further improve their magnetic properties. In this study, we investigated how the interparticle distance (also known as neighbor distance) of SPIO nanocrystals within clusters affect their magnetic relaxation behaviors. To adjust the neighbor distance, different amount of cholesterol (CHO) was chosen as model spacers embedded into SPIO nanocluster systems with the help of amphiphilic diblock copolymer poly(ethylene glyco)-polyester. Small-angle X-ray scattering was applied to quantify the neighbor distance of SPIO clusters. The results demonstrated that the averaged SPIO nanocrystal neighbor distance of nanoclusters increased with higher amount of added CHO. Moreover, these SPIO nanocrystal clusters had the prominent magnetic relaxation properties. Simultaneously, controlling of SPIO nanocrystal neighbor distance can regulate the saturation magnetization (Ms) and magnetic resonance (MR) T2 relaxation of the aggregation, and ultimately obtain better MR contrast effects with decreased neighbor distance.

Control of the interparticle spacing in superparamagnetic iron oxide nanoparticle clusters by surface ligand engineering**Project supported by the National Key Basic Research Program of China (Grant No. 2013CB933903), the National Key Technology R&D Program of China (Grant No. 2012BAI23B08), and the National Natural Science Foundation of China (Grant Nos. 20974065, 51173117, and 50830107).

Polymer-mediated self-assembly of superparamagnetic iron oxide (SPIO) nanoparticles allows modulation of the structure of SPIO nanocrystal cluster and their magnetic properties. In this study, dopamine-functionalized polyesters (DA-polyester) were used to directly control the magnetic nanoparticle spacing and its effect on magnetic resonance relaxation properties of these clusters was investigated. Monodisperse SPIO nanocrystals with different surface coating materials (poly(ε-caprolactone), poly(lactic acid)) of different molecular weights containing dopamine (DA) structure (DA-PCL2k, DA-PCL1k, DA-PLA1k)) were prepared via ligand exchange reaction, and these nanocrystals were encapsulated inside amphiphilic polymer micelles to modulate the SPIO nanocrystal interparticle spacing. Small-angle x-ray scattering (SAXS) was applied to quantify the interparticle spacing of SPIO clusters. The results demonstrated that the tailored magnetic nanoparticle clusters featured controllable interparticle spacing providing directly by the different surface coating of SPIO nanocrystals. Systematic modulation of SPIO nanocrystal interparticle spacing can regulate the saturation magnetization (Ms) and T2 relaxation of the aggregation, and lead to increased magnetic resonance (MR) relaxation properties with decreased interparticle spacing.

Core–shell superparamagnetic iron oxide nanoparticle (SPION) clusters: TEM micrograph analysis, particle design and shape analysis

For the first time, particle shape analysis of silica coated iron oxide (maghemite/magnetite) nanoparticle clusters (core–shell nanostructures) is discussed using computational methods. We analyzed three samples of core–shell nanostructures synthesized with different thickness of the silica shell. A new computational method is presented and successfully applied to the segmentation of the core–shell nanoparticles, as one of the main problems in image analysis of the TEM micrographs. We have introduced the “circularity coefficient”, marked with kcirc and defined as the ratio of circularity measure C2(S) of nanoparticles core and circularity measure core–shell nanoparticles in order to answer the question how the shell affects the overall shape of the final core–shell structure, with respect to circularity. More precisely, the “circularity coefficient” determines whether the circularity of the core–shell nanoparticle is higher, lower or equal to the circularity of the core. We have also determined the shell's share in the overall area of the core–shell nanoparticle. The core–shell nanoparticle clusters here investigated exhibit superparamagnetic properties at room temperature, thus emphasizing their potential for use in practical applications such as in biomedical and particle separation. We show that the saturation magnetization strength can be easily adjusted by controlling the thickness of the silica shell.

Solvothermally Driven Mn Doping and Clustering of Iron Oxide Nanoparticles for Heat Delivery Applications.

Direct interactions between nanoparticles of Mn-doped magnetite or maghemite (clearly differentiated by Raman spectroscopy) grouped in spherical clusters minimize the effect related to their characteristic magnetic dead layer at the surface. Hence, the clustering process jointly with the manganese doping renders these ferrite nanostructures very attractive as displaying increased saturation magnetization, offering, consequently, outstanding values of the specific absorption rate (SAR) for heat delivery. The whole picture for bio-related applications has been considered, with issues related to magnetic manipulation, colloidal stability, and biocompatibility.

Lipase coated clusters of iron oxide nanoparticles for biodiesel synthesis in a solvent free medium

Methyl or ethyl esters of long chain fatty acids are called biodiesel. Biodiesel is synthesized by the alcoholysis of oils/fats. In this work, lipase from Thermomyces lanuginosus was precipitated over the clusters of Fe3O4 nanoparticles. This biocatalyst preparation was used for obtaining biodiesel from soybean oil. After optimization of both immobilization conditions and process parameters, complete conversion to biodiesel was obtained in 3h and on lowering the enzyme amount, as little as 1.7U of lipase gave 96% conversion in 7h. The solvent free media with oil:ethanol (w/w) of 1:4 and 40°C with 2% (w/w) water along with 20% (w/w) silica (for facilitating acyl migration) were employed for reaching this high % of conversion. The biocatalyst design enables one to use a rather small amount of lipase. This should help in switching over to a biobased production of biodiesel.

Magnetic Assembly of Superparamagnetic Iron Oxide Nanoparticle Clusters into Nanochains and Nanobundles

We report on the syntheses of magnetoresponsive, superparamagnetic nanostructures with highly anisotropic shapes, i.e., nanochains of controlled length and their bundles (nanobundles). These nanochains and nanobundles were obtained by the simultaneous magnetic assembly of superparamagnetic nanoparticle clusters (SNCs) and the fixation of the assembled SNCs with an additional layer of deposited silica, produced by a sol-gel process. This low-cost approach provides excellent length control of the short nanochains (approximately 6 or 14 SNCs per nanochain) and fine-tuning of the spacing between the neighboring SNCs inside an individual nanochain. Our magnetically responsive superparamagnetic nanostructures have a controlled aspect ratio, a uniform size, and a well-defined shape, and they express good colloidal stability. This general approach should lead to new, advanced applications of the nanochains and nanobundles in the treatment of cancer and in the ability to magnetically manipulate liquid and photonic crystals.

Nanocluster of superparamagnetic iron oxide nanoparticles coated with poly (dopamine) for magnetic field-targeting, highly sensitive MRI and photothermal cancer therapy

In this paper, a core–shell nanocomposite of clusters of superparamagnetic iron oxide nanoparticles coated with poly(dopamine) (SPION clusters@PDA) is fabricated as a magnetic field-directed theranostic agent that combines the capabilities of highly sensitive magnetic resonance imaging (MRI) and photothermal cancer therapy. The highly concentrated SPION cluster core is suitable for sensitive MRI due to its superparamagnetic properties, and the poly(dopamine) coating layer can induce cancer cell death under near-infrared (NIR) laser irradiation because of the photothermal conversion ability of PDA. MRI scanning reveals that the nanocomposite has relatively high r2 and r2* relaxivities, and the r2* values are nearly threefold higher than the r2 values because of the clustering of the SPIONs in the nanocomposite core. Due to the rapid response to magnetic field gradients, enhanced cellular uptake of our nanocomposite mediated by an external magnetic field can be achieved, thus producing significantly enhanced local photothermal killing efficiency against cancer cells under NIR irritation.

Solvothermal synthesis and characterization of monodisperse superparamagnetic iron oxide nanoparticles

Abstract A series of magnetic iron oxide nanoparticle clusters with different structure guide agents were synthesized by a modified solvothermal method and characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), thermogravimetric analyses (TG), a vibrating sample magnetometer (VSM) and Fourier transform infrared spectroscopy (FTIR). It is found that the superparamagnetic nanoparticles guided by NaCit (sodium citrate) have high saturation magnetization ( M s ) of 69.641 emu/g and low retentivity ( M r ) of 0.8 emu/g. Guiding to form superparamagnetic clusters with size range of 80–110 nm, the adherent small-molecule citrate groups on the surface prevent the prefabricated ferrite crystals growing further. In contrast, the primary small crystal guided and stabilized by the PVP long-chain molecules assemble freely to larger ones and stop growing in size range of 100–150 nm, which has saturation magnetization ( M s ) of 97.979 emu/g and retentivity ( M r ) of 46.323 emu/g. The relevant formation mechanisms of the two types of samples are proposed at the end. The superparamagnetic ferrite clusters guided by sodium citrate are expected to be used for movement controlling of passive interference particles to avoid aggregation and the sample guided by PVP will be a candidate of nanometer wave absorbing material.

Polymer-assisted self-assembly of superparamagnetic iron oxide nanoparticles into well-defined clusters: controlling the collective magnetic properties.

The combination of superstructure-forming amphiphilic block copolymers and superparamagnetic iron oxide nanoparticles produces new nano/microcomposites with unique size-dependent properties. Herein, we demonstrate the controlled clustering of superparamagnetic iron oxide nanoparticles (SPIOs) ranging from discretely encapsulated SPIOs to giant clusters, containing hundreds or even more particles, using an amphiphilic polyisoprene-block-poly(ethylene glycol) diblock copolymer. Within these clusters, the SPIOs interact with each other and show new collective properties, neither obtainable with singly encapsulated nor with the bulk material. We observed cluster-size-dependent magnetic properties, influencing the blocking temperature, the magnetoviscosity of the liquid suspension, and the r2 relaxivity for magnetic iron oxide nanoparticles. The clustering methodology can be expanded also to other nanoparticle materials [CdSe/CdS/ZnS core/shell/shell quantum dots (QDs), CdSe/CdS quantum dots/quantum rods (QDQRs), gold nanoparticles, and mixtures thereof].

High interfacial activity of polymers "grafted through" functionalized iron oxide nanoparticle clusters.

The mechanism by which polymers, when grafted to inorganic nanoparticles, lower the interfacial tension at the oil-water interface is not well understood, despite the great interest in particle stabilized emulsions and foams. A simple and highly versatile free radical "grafting through" technique was used to bond high organic fractions (by weight) of poly(oligo(ethylene oxide) monomethyl ether methacrylate) onto iron oxide clusters, without the need for catalysts. In the resulting ∼1 μm hybrid particles, the inorganic cores and grafting architecture contribute to the high local concentration of grafted polymer chains to the dodecane/water interface to produce low interfacial tensions of only 0.003 w/v % (polymer and particle core). This "critical particle concentration" (CPC) for these hybrid inorganic/polymer amphiphilic particles to lower the interfacial tension by 36 mN/m was over 30-fold lower than the critical micelle concentration of the free polymer (without inorganic cores) to produce nearly the same interfacial tension. The low CPC is favored by the high adsorption energy (∼10(6) kBT) for the large ∼1 μm hybrid particles, the high local polymer concentration on the particles surfaces, and the ability of the deformable hybrid nanocluster cores as well as the polymer chains to conform to the interface. The nanocluster cores also increased the entanglement of the polymer chains in bulk DI water or synthetic seawater, producing a viscosity up to 35,000 cP at 0.01 s(-1), in contrast with only 600 cP for the free polymer. As a consequence of these interfacial and rheological properties, the hybrid particles stabilized oil-in-water emulsions at concentrations as low as 0.01 w/v %, with average drop sizes down to 30 μm. In contrast, the bulk viscosity was low for the free polymer, and it did not stabilize the emulsions. The ability to influence the interfacial activity and rheology of polymers upon grafting them to inorganic particles, including clusters, may be expected to be broadly applicable to stabilization of emulsions and foams.

Diblock‐Copolymer‐Mediated Self‐Assembly of Protein‐Stabilized Iron Oxide Nanoparticle Clusters for Magnetic Resonance Imaging

Superparamagnetic iron oxide nanoparticles (SPIONs) can be used as efficient transverse relaxivity (T2 ) contrast agents in magnetic resonance imaging (MRI). Organizing small (D<10 nm) SPIONs into large assemblies can considerably enhance their relaxivity. However, this assembly process is difficult to control and can easily result in unwanted aggregation and precipitation, which might further lead to lower contrast agent performance. Herein, we present highly stable protein-polymer double-stabilized SPIONs for improving contrast in MRI. We used a cationic-neutral double hydrophilic poly(N-methyl-2-vinyl pyridinium iodide-block-poly(ethylene oxide) diblock copolymer (P2QVP-b-PEO) to mediate the self-assembly of protein-cage-encapsulated iron oxide (γ-Fe2 O3 ) nanoparticles (magnetoferritin) into stable PEO-coated clusters. This approach relies on electrostatic interactions between the cationic N-methyl-2-vinylpyridinium iodide block and magnetoferritin protein cage surface (pI≈4.5) to form a dense core, whereas the neutral ethylene oxide block provides a stabilizing biocompatible shell. Formation of the complexes was studied in aqueous solvent medium with dynamic light scattering (DLS) and cryogenic transmission electron microcopy (cryo-TEM). DLS results indicated that the hydrodynamic diameter (Dh ) of the clusters is approximately 200 nm, and cryo-TEM showed that the clusters have an anisotropic stringlike morphology. MRI studies showed that in the clusters the longitudinal relaxivity (r1 ) is decreased and the transverse relaxivity (r2 ) is increased relative to free magnetoferritin (MF), thus indicating that clusters can provide considerable contrast enhancement.

Toward Design of Magnetic Nanoparticle Clusters Stabilized by Biocompatible Diblock Copolymers for T2-Weighted MRI Contrast

We report the fabrication of magnetic particles comprised of clusters of iron oxide nanoparticles, 7.4 nm mean diameter, stabilized by a biocompatible, amphiphilic diblock copolymer, poly(ethylene oxide-b-D,L-lactide). Particles with quantitative incorporation of up to 40 wt % iron oxide and hydrodynamic sizes in the range of 80-170 nm were prepared. The particles consist of hydrophobically modified iron oxide nanoparticles within the core-forming polylactide block with the poly(ethylene oxide) forming a corona to afford aqueous dispersibility. The transverse relaxivities (r2) increased with average particle size and exceeded 200 s(-1) mM Fe(-1) at 1.4 T and 37 °C for iron oxide loadings above 30 wt %. These experimental relaxivities typically agreed to within 15% with the values predicted using analytical models of transverse relaxivity and cluster (particle core) size distributions derived from cryo-TEM measurements. Our results show that the theoretical models can be used for the rational design of biocompatible MRI contrast agents with tailored compositions and size distributions.

Water transverse relaxation rates in aqueous dispersions of superparamagnetic iron oxide nanoclusters with diverse hydrophilic coating

The article introduces transverse relaxation rates of water protons in the aqueous hydrophilic colloids synthesized on the basis of the oleate-coated iron oxide nanoparticles (17nm) with non-ionic and cationic surfactants, triblock copolymers, polyethyleneimine (PEI) and 4,5-dihydroxybenzene-1,3-disulfoacid disodium salt (Tiron) as hydrophilic components. The IR spectroscopy, atomic force and transmission electron microscopy, along with dynamic light scattering data were obtained to evaluate the content of the hydrophilic coating, the clustering of iron oxide nanoparticles within the hydrophilic covering and the aggregation of hydrophilic nanoclusters. The analysis of the obtained results together with transverse relaxation rates under various concentration, counter-ion and temperature conditions reveals the particular importance of the water molecules diffusion in the outer hydrophilic layer versus the nearest to the iron oxide core layer. The obtained results reveal the counter ion binding with the charged hydrophilic iron oxide colloids as a route to affect the water transverse relaxation rates by biorelevant anionic substrates such as adenosine triphosphates.