Method for determination of peak areas in nonequilibrium capillary electrophoresis of equilibrium mixtures

Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) facilitates determination of both the kinetic constants (k(off)) and the equilibrium constants (K(d)) of complex dissociation from a single experiment. A typical NECEEM electropherogram consists of two peaks and an "exponential bridge" between them, smoothly merging into the peaks. The values of k(off) and K(d) are usually calculated with simple algebraic formulas, by utilizing the areas of the peaks and the bridge. Accurate determination of the two constants requires accurate positioning of the two boundaries separating the bridge from the peaks. Here, we propose a more systematic method for the determination of boundaries between the peaks and the bridge. The method involves a simple geometrical analysis of a NECEEM electropherogram based on an assumption of symmetry in ordinary electrophoretic peaks. To test the method, we (i) constructed a series of computer-simulated NECEEM electropherograms, (ii) determined the two boundaries with our method, and (iii) calculated the values of k(off) and K(d). We found that the deviation of the calculated values from those used to simulate the electropherograms did not exceed 15% for k(off) and 25% for K(d), as long as the peaks and the bridge were visually identifiable. We finally applied the method to the determination of K(d) and k(off) values for the interaction between AlkB protein and its DNA aptamer. The developed method for rational boundary determination in NECEEM will facilitate accurate data analysis in a simple and efficient manner.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures, mathematical model

We recently introduced a new electrophoretic method, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). NECEEM provides a unique way of finding kinetic and equilibrium parameters of the formation of intermolecular complexes from a single electropherogram and allows for the use of weak affinity probes in protein quantitation. In this work, we study theoretical bases of NECEEM by developing a mathematical model for the new method. By solving a system of partial differential equations with diffusion in linear approximation, we found the analytical solution for concentrations of components involved in complex formation as functions of time from the beginning of separation and position in the capillary. The nonnumerical nature of the solution makes it a powerful tool in studying the theoretical foundations of the NECEEM method and modeling experimental results. We demonstrate the use of the model for finding binding parameters of complex formation by nonlinear regression of NECEEM electropherograms obtained experimentally.     

Thermochemistry of protein-DNA interaction studied with temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures

We introduce temperature-controlled nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and demonstrate its use to study thermochemistry of protein-DNA interactions. Being a homogeneous kinetic method, temperature-controlled NECEEM uniquely allows finding temperature dependencies of equilibrium and kinetic parameters of complex formation without the immobilization of the interacting molecules on the surface of a solid substrate. In this work, we applied temperature-controlled NECEEM to study the thermochemistry of two protein-DNA pairs: (i) Taq DNA polymerase with its DNA aptamer and (ii) E. coli single-stranded DNA binding protein with a 20-base-long single-stranded DNA. We determined temperature dependencies of three parameters: the equilibrium binding constant (Kb), the rate constant of complex dissociation (k(off)), and the rate constant of complex formation (k(on)). The Kb(T) functions for both protein-DNA pairs had phase-transition-like points suggesting temperature-dependent conformational changes in structures of the interacting macromolecules. Temperature dependencies of k(on) and k(off) provided insights into how the conformational changes affected two opposite processes: binding and dissociation. Finally, thermodynamic parameters, DeltaH and DeltaS, for complex formation were found for different conformations. With its unique features and potential applicability to other macromolecular interactions, temperature-controlled NECEEM establishes a valuable addition to the arsenal of analytical methods used to study dynamic molecular complexes.     

Selection of smart aptamers by methods of kinetic capillary electrophoresis

We coin the term "smart aptamers" -- aptamers with predefined binding parameters (k(on), k(off), Kd) of aptamer-target interaction. Aptamers, in general, are oligonucleotides, which are capable of binding target molecules with high affinity and selectivity. They are considered as potential therapeutic targets and also thought to rival antibodies in immunoassay-like analyses. Aptamers are selected from combinatorial libraries of oligonucleotides by affinity methods. Until now, technological limitations have precluded the development of smart aptamers. Here, we report on two kinetic capillary electrophoresis techniques applicable to the selection of smart aptamers. Equilibrium capillary electrophoresis of equilibrium mixtures was used to develop aptamers with predefined equilibrium dissociation constants (Kd), while nonequilibrium capillary electrophoresis of equilibrium mixtures facilitated selection of aptamers with different dissociation rate constants (k(off)). Selections were made for MutS protein, for which aptamers have never been previously developed. Both theoretical and practical aspects of smart aptamer development are presented, and the advantages of this new type of affinity probes are described.     

Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-Bbeta as a model system

Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-Bbeta (SH2-Bbeta (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K(d) = 82 +/- 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-Bbeta (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K(d) (101 +/- 12 nM in good agreement with FA measurements) and dissociation rate (k(off) = 0.95 +/- 0.02 s(-)(1) corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K(d) or k(off); however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.     

Comparison of Hadamard transform and signal-averaged detection for microchannel electrophoresis

A Hadamard transform (HT) detection method for microchip capillary electrophoresis with laser-induced fluorescence and a charge-coupled device (CCD) is described and compared to signal-averaged detection. A low-noise CCD camera is used to image a section of a separation channel where each camera pixel can be thought of as a unique detector. For signal averaging, electropherograms corresponding to individual pixels can be averaged for improved S/N. HT detection is performed on each pixel electropherogram to generate a contour plot electropherogram. The multiple injections required for HT provides an enhancement at the cost of longer times for the pseudorandom injection sequences. A short sample injection length of 0.25 s is used to reduce the overall analysis time and improve sensitivity compared to previously published results. An injection sequence is performed on the microchip that is based on a cyclic S-matrix of 513 elements that generates an 8-fold improvement in S/N compared to a single injection. This spatially resolved HT detection method is also capable of performing a multicomponent separation. Signal-averaged HT and single-injection data are compared to experimental HT and single-injection results. The unique capabilities of each method are described.     

A facile synthesis and physical properties of nano-sized dendritic alpha,epsilon-poly(L-lysine)s for the delivery of nucleic acids

A series of nano-sized dendritic alpha,epsilon-poly(L-lysine)s (DPL) were synthesized by the solid-phase peptide synthesis method, using a core epsilon-peptide structure consisting of eight lysine residues. Surface amines of dendritic alpha,epsilon-poly(L-lysine) were characterized by comparing the retention times of a reverse phase HPLC with the electrophoretic mobilities of capillary zone electrophoresis (CZE) and non-denatured polyacrylamide gel electrophoresis (PAGE). The elution times of alpha,epsilon-poly(Llysine) in HPLC were well correlated with the electrophoretic mobilities of CZE and PAGE. The separation was dependent on size, shape of molecule and the number of surface amine. The alpha,epsilon-poly(L-lysine) formed a complex with nucleic acids at various charge ratios and the degree of complex formation was size- and structure-dependent. Atomic force microscopy of the complex visualized the size and morphology of alpha,epsilon-poly(L-lysine)/DNA complex as a nano-sized spherical shape. The small size in complex formation provides a promise for in vivo therapeutic application of dendritic alpha,epsilon-poly(L-lysine)s or their derivatives in the delivery of gene or oligonucleotide.     

Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis

Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, and their fluorescence emission is monitored. We prove theoretically that the power spectrum of fluctuations in the fluorescence intensity gives a virtual electropherogram. The profile of the electropherogram and the number of theoretical plates are in general obtained by using analytical methods. Characterizing the capillary length within the excitation beams as the effective length, we compare CE/ patterned FCS with conventional CE. Numerical simulations on capillary gel electrophoresis of DNA predict that the optimized CE/patterned FCS is superior to conventional CE when the effective length is shorter than 1 cm. The experimental feasibility of this technique is demonstrated in the fluorometry of TOTO-1-stained DNA. For an effective length of 740 microm, a maximum number of plates of 7400, and a resolution of 1.0 were obtained with a one-component injection of pUC18 DNA and a two-component injection of pUC 18 DNA and lambda DNA, respectively.     

Two-beam fluorescence cross-correlation spectroscopy in an electrophoretic mobility shift assay

Two-beam fluorescence cross-correlation spectroscopy (FCCS) was used to resolve the bound and unbound fractions of fluorescently labeled single-stranded DNA (ssDNA) in a ssDNA-protein complex as the analyte solution flowed continuously through an electrophoresis capillary. Cross-correlation of the single molecule fluorescence from two spatially separate excitation laser beams resulted in cross-correlation functions that consisted of well-resolved peaks characteristic of the different electrophoretic flow velocities of the bound and unbound ssDNA. This decoupled the molecular parameters of the bound and unbound ssDNA used to model the cross-correlation function, which enabled the relative concentrations to be determined without prior knowledge of the pure-component cross-correlation functions, as would be required in an analogous autocorrelation analysis. The relative concentrations of the bound and unbound ssDNA were determined by two-beam FCCS within 2-6% precision, even for samples that contained as little as 5% unbound ssDNA, and were consistent with the results obtained by capillary electrophoresis (CE) separation of the same samples. Data sufficient to obtain these results was acquired in 10-15 s per sample. Fluorescently labeled poly(dT)39 complexed with the single stranded DNA binding protein of Escherichia coli served as the model system. The measured dissociation constant of 2.5+/-0.9 nM agreed with the literature value for this complex within experimental error. The CE/two-beam FCCS experiment described here is part of a family of techniques that use single molecule fluorescence detection to resolve different components in an electrophoresis system. Advantages of these methods relative to separations-based CE include enhanced sensitivity, the potential for higher speed analyses, elimination of the sample plug injection step, and the ability to carry out the analysis in shorter flow channels.     

High-throughput,high-sensitivity genetic mutation detection by tandem single-strand conformation polymorphism/heteroduplex analysis capillary array electrophoresis

We present the first optimization of linear polyacrylamide (LPA)-based DNA separation matrixes for an automated tandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method, implemented in capillary arrays dynamically coated with poly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed both SSCP and HA species to be produced in one step in a single tube and distinguished in a single electrophoretic analysis. A simple, two-color fluorescent sample labeling and detection strategy enabled unambiguous identification of all DNA species in the electropherogram, both single- and double-stranded. Using these protocols and a panel of 11 p53 mutant DNA samples in comparison with wild-type, we employed high-throughput capillary array electrophoresis (CAE) to carry out a systematic and simultaneous optimization of LPA weight-average molar mass (Mw) and concentration for SSCP/HA peak separation. The combination of the optimized LPA matrix (6% LPA, Mw 600 kDa) and a hydrophilic, adsorbed polyDuramide wall coating was found to be essential for resolution of CAE-SSCP/HA peaks and yielded sensitive mutation detection in all 11 p53 samples initially studied. A larger set of 32 mutant DNA specimens was then analyzed using these optimized tandem CAE-SSCP/HA protocols and materials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA). This simple, highly sensitive tandem SSCP/HA mutation detection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to the ease of the capillary coating protocol and the low viscosity of the matrix.     

Using nonequilibrium capillary electrophoresis of equilibrium mixtures for the determination of temperature in capillary electrophoresis

Until now, all methods for temperature sensing in capillary electrophoresis (CE) relied on molecular probes with temperature-dependent spectral/optical properties. Here we introduce a nonspectroscopic approach to determining temperature in CE. It is based on measuring a temperature-dependent rate constant of complex dissociation by means of a kinetic CE method known as nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). Conceptually, a calibration curve of "the rate constant versus temperature" is built using NECEEM and a CE instrument with a reliable temperature control or, alternatively, a nonelectrophoretic method, such as surface plasmon resonance. The calibration curve is then used to find the temperature during CE in the same buffer but with another CE apparatus or under otherwise different conditions (cooling efficiency, length and diameter of the capillary, electrical field, etc.). In this proof-of-principle work, we used the dissociation of a protein-DNA complex to demonstrate that the NECEEM-based temperature determination method allows for temperature determination in CE with a precision of 2 degrees C. Then, we applied the NECEEM-based temperature determination method to study heat dissipation efficiency in CE instruments with active and passive cooling of the capillary. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, e.g. electrochemical detection. A "kinetic probe" can be coloaded into the capillary along with a sample for in situ temperature measurements. Higher order chemical reactions can also be used for temperature sensing, provided a kinetic CE method for measuring a corresponding rate constant is available.     

Hadamard transform capillary electrophoresis combined with absorption spectrometry

A novel injection device for applying absorption spectrometry to Hadamard transform (HT) capillary electrophoresis is described. A small hole, at the center part of the capillary, functions as an inlet port for the sample. The hole is immersed in a sample solution and the end of the capillary that is usually employed for sample introduction is immersed in a buffer solution. An ultraviolet absorption detector is placed between the sample injection port and the other end of the capillary filled with a buffer solution. A high potential is continuously applied between the injection port and the end of the capillary, which allows the sample solution to be introduced into the separation capillary. By application of a higher potential modulated according to a Hadamard code between both ends of the capillary, the buffer solution is injected into the separation capillary. In some preliminary experiments, this injection device was utilized to introduce a single sample segment into a capillary. As expected, a single peak was observed in the electropherogram for a sample containing a single component. This device was then employed for multiple sample injection in HT capillary electrophoresis. An 8-fold improvement in the S/N ratio was observed when the HT technique was used, in which a 255-order of a Hadamard matrix was used, as expected from theory. The present approach was also utilized for the sensitive detection of a sample comprised of multiple components.     

Revealing equilibrium and rate constants of weak and fast noncovalent interactions

Rate and equilibrium constants of weak noncovalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine k(on), k(off), and K(d) of weak (K(d) > 1 μM) and fast kinetics (relaxation time, τ < 0.1 s) in quasi-equilibrium for multiple unlabeled ligands simultaneously in one microreactor. Conceptually, an equilibrium mixture (EM) of a ligand (L), target (T), and a complex (C) is prepared. The mixture is introduced into the beginning of a capillary reactor with aspect ratio >1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. The combination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is a function of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shape (width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplex determination of kinetic parameters describing weak (3 mM > K(d) > 80 μM) and fast (0.25 s ≥ τ ≥ 0.9 ms) noncovalent interactions between four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and α- and β-cyclodextrins. The affinity of the drugs was significantly higher for β-cyclodextrin than α-cyclodextrin and mostly determined by the rate constant of complex formation.     

Method for the elimination of chromatographic bias from measured capillary electrophoretic effective mobility values

A method based on a modified version of pressure-mediated capillary electrophoresis (PreMCE) has been developed for the elimination of the chromatographic bias inherent in effective electrophoretic mobilities measured by capillary electrophoresis. This new five-band PreMCE method can be readily executed on most commercial capillary electrophoresis instruments. It yields not only precise but also accurate effective mobilities and electroosmotic flow rates, even when the analytes and electroosmotic flow markers are strongly retained on the coated fused-silica capillary wall.     

Vacancy capillary zone electrophoresis and differential capillary zone electrophoresis: variations on a well-known theme

Two new variants on capillary zone electrophoresis (CZE) are described and experimentally investigated. In vacancy CZE the sample, diluted with a background electrolyte, fills the electrode vessels and the separation capillary. When pure background electrolyte is injected, the resulting electropherogram represents the composition of the sample. The electropherogram is almost identical with the result of a conventional CZE experiment. In differential CZE the sample again fills the electrode vessels and the separation compartment. After injection of a slightly different sample, a differential electropherogram is obtained that represents the differences between the two samples. The retention times of both new variants are comparable with conventional CZE, the separation efficiency, in terms of theoretical plates, is marginally lower, and both show good quantitative characteristics. The concept of vacancy electrophoresis explains the origin of unexpected system peaks in conventional CZE when multiple co-ions in the background electrolyte are used.     

Two-dimensional fluorescence correlation in capillary electrophoresis for peak resolution and species identification

A new spectroscopic dimension-fluorescence intensity correlation--is introduced to enhance peak resolution and species identification in capillary electrophoresis. In two-dimensional correlation CE, a conventional electropherogram is spread into two dimensions through cross-correlation analysis of fluorescence time response. A laser that is sinusoidally modulated in intensity is used as the excitation source. Three channels of information are collected during a CE run: the steady-state intensity, the ac amplitude, and the phase-resolved fluorescence intensity. The correlation between two chosen channels is then evaluated. A two-dimensional correlation electropherogram consists of a plot of the correlation intensity versus two axes of migration time. Through correlation analysis, species discrimination and peak resolution are significantly enhanced without having to physically separate the solutes. Two-dimensional correlation CE showed complete resolution between two overlapping sample peaks with a resolution of 0.28 in the conventional one-dimensional electropherogram. In separations of polycyclic aromatic hydrocarbons by micellar electrokinetic chromatography (MEKC), two-dimensional correlation analysis resolved all overlapping elution peaks unseparable by one-dimensional MEKC, demonstrating the utility of 2D correlation in separation method development. The capability of 2D correlation CE in species identification is demonstrated with a sequence of 39 consecutively injected peaks containing four fluorescent dyes. Species identification in sequencing is achieved without complex data treatment in two-dimensional correlation CE.     

A sheathless nanoflow electrospray interface for on-line capillary electrophoresis mass spectrometry

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of low-femtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.     

Homogeneous immunoassay for detection of TNT and its analogues on a microfabricated capillary electrophoresis chip

A homogeneous immunoassay for TNT and its analogues is developed using a microfabricated capillary electrophoresis chip. The assay is based on the rapid electrophoretic separation of an equilibrated mixture of an anti-TNT antibody, fluorescein-labeled TNT, and unlabeled TNT or its analogue. The band intensities of the free fluorescein-labeled TNT and of the antibody-antigen complex reveal the relative equilibrated concentrations. Titration of the anti-TNT antibody with a fluorescein-labeled TNT derivative yields a binding constant of (3.9 +/- 1.3) x 10(9) M(-1). The dissociation rate constant of the complex is determined by kinetic capillary electrophoresis using a folded channel and a rotary scanner to interrogate the separation at multiple time points. The dissociation rate constant is found to be 0.035 +/- 0.005 s(-1), and the resulting binding rate constant is (1.4 +/- 0.7) x 10(7) M(-1) s(-1). Binding constants of TNT and five of its analogues are determined by competitive assays: TNT (4.3 +/- 2.6) x 10(8) M(-1); 1,3,5-trinitrobenzene (5.1 +/- 3.3) x 10(7) M(-1); picric acid (7.5 +/- 4.4) x 10(6) M(-1); 2,4-dinitrotoluene (7.9 +/- 4.0) x 10(6) M(-1); 1,3-dinitrobenzene (1.0 +/- 0.7) x 10(6) M(-1); and 2,4-dinitrophenol (5.1 +/- 3.0) x 10(4) M(-1). TNT and its analogues can be assayed with high sensitivity (LOD 1 ng/mL) and with a wide dynamic range (1-300 ng/mL) using this chip-based method.     

Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures

We propose a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analyses of proteins. The method is based on nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of a protein with its fluorescently labeled aptamer. In general, NECEEM of a protein with a fluorescently labeled aptamer generates an electropherogram with three characteristic features: two peaks and an exponential curve. Two peaks correspond to (i) the equilibrium amount of free aptamer in the equilibrium mixture and (ii) the amount of the protein-aptamer complex that remains intact at the time of detection. The exponential part is ascribed to the complex decaying during separation under nonequilibrium conditions. Simple analysis of the three features in experiments with known concentrations of the protein can be used for the determination of the equilibrium dissociation constant, Kd, of the aptamer-protein complex. Similar analysis of the three features in the experiment with unknown concentration of the protein and known Kd value allows the determination of the protein concentration. In this proof-of-principle work, the NECEEM method was applied to the analysis of thrombin using a fluorescein-labeled aptamer under the conditions at which the protein-aptamer complex completely decayed during the separation. We demonstrated that, despite the decay, as few as 4 x 10(6) molecules of the protein could be detected with NECEEM without sacrificing the accuracy. This sensitivity is comparable with that reported by others for the aptamer-based equilibrium method. Thus, the proposed NECEEM-based method allows the use of aptamers for highly sensitive affinity analysis of proteins even when protein-aptamer complexes are unstable.     

Surface-enhanced Raman spectroscopy of chernozem humic acid and their fractions obtained by coupled size exclusion chromatography-polyacrylamide gel electrophoresis (SEC-PAGE)

A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and C+D, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and C+D and, in turn, fractions with lower MS (B and C+D) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.