Amphiphilic Janus Gold Nanoparticles Prepared by Interface‐Directed Self‐Assembly: Synthesis and Self‐Assembly

Materials with Janus structures are attractive for wide applications in materials science. Although extensive efforts in the synthesis of Janus particles have been reported, the synthesis of sub‐10 nm Janus nanoparticles is still challenging. Herein, the synthesis of Janus gold nanoparticles (AuNPs) based on interface‐directed self‐assembly is reported. Polystyrene (PS) colloidal particles with AuNPs on the surface were prepared by interface‐directed self‐assembly, and the colloidal particles were used as templates for the synthesis of Janus AuNPs. To prepare colloidal particles, thiol‐terminated polystyrene (PS‐SH) was dissolved in toluene and citrate‐stabilized AuNPs were dispersed in aqueous solution. Upon mixing the two solutions, PS‐SH chains were grafted to the surface of AuNPs and amphiphilic AuNPs were formed at the liquid–liquid interface. PS colloidal particles decorated with AuNPs on the surfaces were prepared by adding the emulsion to excess methanol. On the surface, AuNPs were partially embedded in the colloidal particles. The outer regions of the AuNPs were exposed to the solution and were functionalized through the grafting of atom‐transfer radical polymerization (ATRP) initiator. Poly[2‐(dimethamino)ethyl methacrylate] (PDMAEMA) on AuNPs were prepared by surface‐initiated ATRP. After centrifugation and dissolving the colloidal particles in tetrahydrofuran (THF), Janus AuNPs with PS and PDMAEMA on two hemispheres were obtained. In acidic pH, Janus AuNPs are amphiphilic and are able to emulsify oil droplets in water; in basic pH, the Janus AuNPs are hydrophobic. In mixtures of THF/methanol at a volume ratio of 1:5, the Janus AuNPs self‐assemble into bilayer structures with collapsed PS in the interiors and solvated PDMAEMA at the exteriors of the structures.     

Interface-directed self-assembly of gold nanoparticles and fabrication of hybrid hollow capsules by interfacial cross-linking polymerization

Amphiphilic gold nanoparticles (AuNPs) were produced at liquid-liquid interface via ligand exchange between hydrophilic AuNPs and disulfide-containing polymer chains. By using oil droplets as templates, hybrid hollow capsules with AuNPs on the surfaces were obtained after interfacial cross-linking polymerization. The volume ratio of toluene to water exerts an important effect on the size of capsules. The average size of the capsules increases with the volume ratio. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used to characterize the hollow structures. In this research, not only one-component but also multicomponent hollow capsules were prepared by copolymerization of acrylamide and hybrid AuNPs at liquid-liquid interface. Because of the improvement in hydrophilicity of the hollow capsules, the average size of multicomponent capsules is bigger than one-component ones in aqueous solution.     

Structure and Stability of Gold Nanoparticles Synthesized Using <Emphasis Type="Italic">Schinus molle</Emphasis> L. Extract

In this work, we exhibited the results of the green synthesis of gold nanoparticles by aqueous extract of Schinus molle L. leaves. The chemical reaction was carried out by varying the plant extract/precursor salt ratio concentration in the aqueous solution. The structural characterization of the nanoparticles was performed using X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). XRD analysis showed that the as-synthesized AuNPs have a face-centered cubic structure. SEM and TEM observations indicated that most of the obtained particles have multiple twinning structures (MTP). The synthesized Au-MTP have particle sizes in the range of 10–60 nm, most of them with an average size of about 24 nm. However, triangular Au plate particles were also obtained, having an average size of 180 nm. Fourier transforms infrared spectroscopy and shows that the functional groups responsible for the chemical reduction of AuNPs are phenolic compounds present in the S. molle L. leaf.     

Effect of Latent Heat in Boiling Water on the Synthesis of Gold Nanoparticles of Different Sizes by using the Turkevich Method

The Turkevich method, involving the reduction of HAuCl₄ with citrate in boiling water, allows the facile production of monodisperse, quasispherical gold nanoparticles (AuNPs). Although, it is well‐known that the size of the AuNPs obtained with the same recipe vary slightly (as little as approximately 4 nm), but noticeably, from one report to another, it has rarely been studied. The present work demonstrates that this size variation can be reconciled by the small, but noticeable, effect that the latent heat in boiling water has on the size of the AuNPs obtained by using the Turkevich method. The increase in latent heat during water boiling caused an approximately 3 nm reduction in the size of the as‐prepared AuNPs; this reduction in size is mainly a result of accelerated nucleation driven by the extra heat. It was further demonstrated that, the heating temperature can be utilized as an additional measure to adjust the growth rate of AuNPs during the reduction of HAuCl₄ with citrate in boiling water. Therefore, the latent heat of boiling solvents may provide one way to control nucleation and growth in the synthesis of monodisperse nanoparticles.     

Gold nanoparticles prepared by glycinate ionic liquid assisted multi-photon photoreduction

We have successfully prepared gold nanoparticles (AuNPs) with flower-like and spherical morphology through multi-photon photoreduction (MPR) of an aqueous solution of HAuCl(4) and (2-hydroxyethyl) trimethylammonium glycinate ([HETMA][Gly]) ionic liquid (IL) through the use of a femtosecond laser. The results of (1)H NMR and UV-Vis absorption indicated that AuNPs were produced from the photoreduction of the [Gly]-Au(iii) complex. Spherical AuNPs of about 2.5 nm were obtained on the solution when irradiated for 2 h, then aggregated into flower-like AuNPs of several tens of nanometers assisted by the IL with an increase in the irradiation time. Furthermore, precipitates of spherical AuNPs with the size of around 15 nm were formed after being irradiated for 6 h. The mechanisms of the MPR reaction and controlled growth of AuNPs have also been discussed.     

Synthesis and investigation of dual pH‐ and temperature‐responsive behaviour of poly[2‐(dimethylamino)ethyl methacrylate]‐grafted gold nanoparticles

Gold nanoparticles (AuNPs) were synthesized by reduction of chloroauric acid (HAuCl₄) aqueous solution with hydrazine monohydrate. The AuNPs were immediately treated with cysteamine to obtain amine‐functionalized nanoparticles (Au‐NH₂). The reaction of Au‐NH₂ with epichlorohydrin and subsequent treatment with sodium hydroxide gave epoxidized AuNPs (Au‐EP). Then, thiol‐capped AuNPs (Au‐SH) were synthesized by reaction of Au‐EP with cysteamine. A ‘grafting to’ approach was utilized to graft bromine‐terminated poly(N ,N ′‐dimethylaminoethyl methacrylate), synthesized via aqueous atom transfer radical polymerization, with various molecular weights (6280, 25 800, 64 200 and 87 600 g mol⁻¹) onto Au‐SH to obtain Au‐P1, Au‐P2, Au‐P3 and Au‐P4 samples, respectively. All samples were exposed to temperature and pH variations, and Z‐average diameter was monitored using dynamic light scattering. According to the results, polymer‐grafted nanoparticles collapsed at lower temperatures with increasing solution pH for all molecular weight ranges due to deprotonation of tertiary amine groups. However, higher molecular weight polymers were more sensitive to pH variation especially in alkaline media. Also, a high degree of agglomeration was observed for Au‐P4 nanoparticles in alkaline media on increasing the temperature to 55 and 65 °C.     

Controllable synthesis of water-soluble gold nanoparticles and their applications in electrocatalysis and surface-enhanced Raman scattering

We report a facile method to synthesize water-soluble gold nanoparticles (AuNPs) using a biosurfactant sodium cholate as reducing reagents and protective groups in aqueous solution at ambient temperature. The diameters (13-70 nm) of uniform AuNPs can be readily adjusted by changing the initial molar ratio of sodium cholate to chloroauric acid (HAuCl(4)). Also, the alkaline condition of preparative solution is found to affect the size of as-synthesized AuNPs. This synthetic approach is one-step and "green". The obtained AuNPs exhibit a good electrocatalytic activity toward methanol oxidation. Meanwhile, the AuNPs thin films can serve as an efficient substrate for surface-enhanced Raman scattering (SERS). Furthermore, platinum nanoparticles (PtNPs) are also prepared by reducing sodium tetrachloro platinate hydrate with sodium cholate.     

Comparative study of amphiphilic hyperbranched and linear polymer stabilized organo‐soluble gold nanoparticles as efficient recyclable catalysts in the biphasic reduction of 4‐nitrophenol

A series of organo‐soluble spherical gold nanoparticles (AuNPs) were prepared through the reduction of HAuCl₄ by NaBH₄ in the presence of amphiphilic hyperbranched polymers that had a hydrophilic hyperbranched polyethylenimine core and a hydrophobic shell formed by many palmitamide (C16) chains. For comparison, the corresponding linear polymeric analog derived from linear polyethylenimine was also used to prepare the organo‐soluble AuNPs. The obtained AuNPs were characterized by transmission electron microscopy. It was found that higher feed ratio of polymer to HAuCl₄ and utilization of polymers with higher C16 density usually resulted in smaller AuNPs with relatively lower polydispersity. Except of the polymer having the pronounced low molecular weight, the molecular weight and the morphology of the amphiphilic polymers had almost no obvious effect on the size of the formed AuNPs. These organo‐soluble AuNPs could be used as efficient catalysts for the biphasic catalytic reduction of 4‐nitrophenol by NaBH₄. Their apparent rate coefficients had correlation with the molecular weight of the used amphiphilic polymers, but were less relevant to the morphology of these polymers. These organo‐soluble AuNPs could be conveniently recovered and reused many times. The morphology of the capping polymers had obvious effect on the lifetime of the AuNPs catalysts in the catalytic reduction of 4‐nitrophenol. Except of the pronounced low molecular weight hyperbranched polymer, the other hyperbranched ones with relatively high molecular weight rendered the AuNPs to have bigger turnover number values than their linear analog. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Surface chemical states of gold nanoparticles prepared using the solution-plasma method in a CsCl aqueous solution

In this study, gold nanoparticles (AuNPs) prepared in a 5 mM CsCl aqueous solution using the solution-plasma method are characterized via transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy with synchrotron radiation (SR-XPS). The particle diameter is measured over the process time via TEM. During the solution-plasma process, small particles of 2.1 to 2.2-nm diameter are generated in the CsCl aqueous solution; these particles then enlarge via Ostwald ripening over time until they reach an equilibrium size of ~13 nm after 36 days. In addition, the surface chemical states of the AuNPs are characterized at different depths via SR-XPS. The SR-XPS measurements obtained using incident X-ray energy (hν) of 945.0 eV revealed that Cs─Au, Cl─Au, and Cs─Cl─Au bonds are present 1.2 nm below the surface. The measurements obtained at an incident X-ray energy of 2515.0 eV showed that Cs─Cl─Au bonding is also present 2.5 nm below the surface, indicating that Cs and Cl strongly interact with Au. The TEM and SR-XPS measurements revealed that 2 processes occur cyclically during the growth process via Ostwald ripening: (i) the Cs and Cl in the aqueous solution adsorb on the AuNP surface and (ii) Au atoms subsequently bond to the AuNPs surface.     

Facile Synthesis of Quasi‐One‐Dimensional Au/PtAu Heterojunction Nanotubes and Their Application as Catalysts in an Oxygen‐Reduction Reaction

An intermediate‐template‐directed method has been developed for the synthesis of quasi‐one‐dimensional Au/PtAu heterojunction nanotubes by the heterogeneous nucleation and growth of Au on Te/Pt core–shell nanostructures in aqueous solution. The synthesized porous Au/PtAu bimetallic nanotubes (PABNTs) consist of porous tubular framework and attached Au nanoparticles (AuNPs). The reaction intermediates played an important role in the preparation, which fabricated the framework and provided a localized reducing agent for the reduction of the Au and Pt precursors. The Pt₇Au PABNTs showed higher electrocatalytic activity and durability in the oxygen‐reduction reaction (ORR) in 0.1 M HClO₄ than porous Pt nanotubes (PtNTs) and commercially available Pt/C. The mass activity of PABNTs was 218 % that of commercial Pt/C after an accelerated durability test. This study demonstrates the potential of PABNTs as highly efficient electrocatalysts. In addition, this method provides a facile strategy for the synthesis of desirable hetero‐nanostructures with controlled size and shape by utilizing an intermediate template.     

Use of alkaline or enzymatic sample pretreatment prior to characterization of gold nanoparticles in animal tissue by single-particle ICPMS

Inductively coupled plasma mass spectrometry in single-particle mode (spICPMS) is a promising method for the detection of metal-containing nanoparticles (NPs) and the quantification of their size and number concentration. Whereas existing studies mainly focus on NPs suspended in aqueous matrices, not much is known about the applicability of spICPMS for determination of NPs in complex matrices such as biological tissues. In the present study, alkaline and enzymatic treatments were applied to solubilize spleen samples from rats, which had been administered 60-nm gold nanoparticles (AuNPs) intravenously. The results showed that similar size distributions of AuNPs were obtained independent of the sample preparation method used. Furthermore, the quantitative results for AuNP mass concentration obtained with spICPMS following alkaline sample pretreatment coincided with results for total gold concentration obtained by conventional ICPMS analysis of acid-digested tissue. The recovery of AuNPs from enzymatically digested tissue, however, was approximately four times lower. Spiking experiments of blank spleen samples with AuNPs showed that the lower recovery was caused by an inferior transport efficiency of AuNPs in the presence of enzymatically digested tissue residues.     

Gold Nanoparticles Carrying Diatomic Molecules (O2 and CO) in Aqueous Solution

Gold nanoparticles (AuNPs) prepared by citrate reduction of aurochloric acid (HAuCl₄) were functionalized by tris(4‐sulfonatophenyl)porphinatoiron(III) (Fe^IIIP2) and poly(ethylene glycol) with thiolated arms (PEG‐SH). Fe^IIIP2 on the AuNP surface existed as its μ‐oxo dimer, which was reduced by Na₂S₂O₄ to yield monomeric Fe^IIP2. Fe^IIP2‐bearing AuNPs were further functionalized through inclusion of two sulfonatophenyl groups of Fe^IIP2 by a per‐O‐methylated β‐cyclodextrin dimer with a pyridine linker (Py3CD) to obtain AuNPs capable of carrying diatomic molecules in the body. The resulting AuNPs (hemoCD‐AuNPs) bound O₂ as well as CO in an aqueous solution. Although a noncolloidal 1:1 complex of 5,10,15,20‐tetrakis(4‐sulfonatophenyl)porphinatoiron(II) and Py3CD injected into the femoral vein of a rat was rapidly excreted in the urine, no excretion was observed with ferric hemoCD‐AuNPs, which were gradually accumulated in the spleen and liver of a rat. These results suggest that hemoCD‐AuNPs can be used as a carrier of diatomic molecules such as O₂ and CO in vivo.     

Facile green synthesis of gold nanoparticles using leaf extract of antidiabetic potent Cassia auriculata

A simple biological method for the synthesis of gold nanoparticles (AuNPs) using Cassia auriculata aqueous leaf extract has been carried out in the present study. The reduction of auric chloride led to the formation of AuNPs within 10 min at room temperature (28°C), suggesting a higher reaction rate than chemical methods involved in the synthesis. The size, shape and elemental analysis were carried out using X-ray diffraction, TEM, SEM-EDAX, FT-IR and visible absorption spectroscopy. Stable, triangular and spherical crystalline AuNPs with well-defined dimensions of average size of 15-25 nm were synthesized using C. auriculata. Effect of pH was also studied to check the stability of AuNPs. The main aim of the investigation is to synthesize AuNPs using antidiabetic potent medicinal plant. The stabilizing and reducing molecules of nanoparticles may promote anti-hyperglycemic if tested further.     

Preparation of Cross‐Linked Micelles from Glycidyl Methacrylate Based Block Copolymers and Their Usages as Nanoreactors in the Preparation of Gold Nanoparticles

This study reports the synthesis of poly(ethylene glycol)methyl ether‐block‐poly(glycidyl methacrylate) (MPEG‐b‐PGMA) diblock, and poly(ethylene glycol)methyl ether‐block‐poly(glycidyl methacrylate)‐block‐poly(methyl methacrylate) (MPEG‐b‐PGMA‐b‐PMMA) triblock copolymers via atom transfer radical polymerization and their self‐assembly behaviors in aqueous media by using acetone as cosolvent. These block copolymers formed near monodisperse core–shell micelles having cross‐linkable cores. Two types of cross‐linked micelles, namely spherical MPEG‐b‐PGMA core cross‐linked (CCL) micelles and MPEG‐b‐PGMA‐b‐PMMA interlayer cross‐linked (ILCL) micelles, were also successfully prepared from these block copolymers by using various bifunctional cross‐linkers such as hexamethylenediamine (HMDA), ethylenediamine (EDA), and 2‐aminoethanethiol (AET). Cross‐linking was successfully carried out via ring‐opening reactions of epoxy residues of hydrophobic‐cores with primary amine or thiol groups of bifunctional cross‐linkers. Finally, these cross‐linked micelles were successfully used as nanoreactors in the synthesis of gold nanoparticles (AuNPs) in aqueous media. Both CCL and ILCL micelles were found to be good stabilizers for AuNPs in aqueous media. Both CCL‐ and ILCL‐stabilized AuNP dispersions were stable for a long time without any size changes and flocculation at room temperature. These cross‐linked stabilized AuNPs exhibited good catalytic activities in the reduction of p‐nitrophenol. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 514–526.     

Synthesis of gold nanoparticle necklaces using linear-dendritic copolymers

Linear-dendritic copolymers containing hyperbranched poly(citric acid) and linear poly(ethylene glycol) blocks (PCA-PEG-PCA) were used as reducing and capping agents to synthesize and support gold nanoparticles (AuNPs). PCA-PEG-PCA copolymers with 1758, 1889 and 3446 molecular weights, called A1, A2 and A3 through this work, respectively, were synthesized using 2, 5, and 10 citric acid/PEG molar ratios. The diameter of A1, A2 and A3 in a fresh water solution was investigated using dynamic light scattering (DLS) and it was between 1.8 and 2.8 nm. AuNPs were simply synthesized and supported by addition a boiling aqueous solution of HAuCl₄ to aqueous solutions of A1, A2 and A3. Supported AuNPs were stable in water for several months and agglomeration was not occurred. The loading capacity of A1, A2 and A3 and the size of synthesized AuNPs were investigated using UV spectroscopy and transmission electron microscopy (TEM). It was found that the loading capacity of PCA-PEG-PCA copolymers depend on the concentration of copolymers and the size of their poly(citric acid) parts directly. For example average loading capacities for 400 μM concentration of A1, A2 and A3 were 32.24, 37.4 and 41.52 μM, respectively, and average loading capacities for 400, 200 and 100 μM concentration of A1 were 32.24, 20.28 and 9.1 μM, respectively. Interestingly there was a reverse relation between the size of synthesized AuNPs and size of poly(citric acid) parts of PCA-PEG-PCA copolymers.     

Effect of surface modification with various thiol compounds on colloidal stability of gold nanoparticles

Gold nanoparticles (AuNPs) are attractive materials due to their special optical and electronic properties. However, they tend to aggregate particularly in the presence of thiol‐containing compounds. In this study, to investigate the effect of surface conjugation with thiol‐containing compounds on colloidal stability, thiol compounds with various structures as modifying agents were used. To this end, AuNPs were synthesized and stabilized by trisodium citrate in aqueous solution, and then modified with thiol‐containing compounds, namely cysteamine hydrochloride (MEA, containing primary amine groups), 2‐mercaptoethanol (BME, containing hydroxyl groups), 1‐dodecanthiol (LCA, containing long‐chain alkyl groups) and thioglycolic acid (TGA, containing carboxylic acid groups). We studied the effect of thiol ligands on solution stability of colloidal AuNPs and on the formation of aggregates originating from the modification process using UV–visible spectroscopy, dynamic light scattering, field emission scanning electron microscopy and transmission electron microscopy. Results showed that surface modification with MEA, BME and LCA led to the formation of aggregates. However, conjugation with TGA showed a concentration‐dependent behaviour: surface modification with low concentration resulted in the formation of aggregates whereas that with high concentration of TGA did not disturb the colloidal stability of AuNPs. Finally, the effect of surface modification on temperature increase of solutions originating from infrared light irradiation was studied, where the temperature increase depends on the surface‐modifying compound.     

Catalytic Reduction of p‐Nitrophenol and Hexacyanoferrate (III) by Borohydride Using Green Synthesized Gold Nanoparticles

A simple and green approach for the synthesis of well‐stabilized gold nanoparticles (AuNPs) using gum Acacia (GA) is presented here. The gum acacia acts as the reductant and stabilizer. The synthesized gold nanoparticles were characterized by using ultraviolet visible (UV‐Vis), fourier transform infrared spectroscopy (FTIR), x‐ray diffraction (XRD), dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. The UV‐Vis study revealed a distinct surface plasmon resonance at 520 – 550 nm, due to the formation of AuNPs. FTIR analysis showed the evidence that –OH groups present in the gum matrix were responsible in reducing the tetra chloroauric acid into AuNPs. XRD studies confirmed the formation of well crystalline nanoparticles with fcc structure and the particle size ranges from 4 – 29 nm, as indicated by TEM analysis. The synthesized gold nanoparticles exhibited homogeneous catalytic activity. The two model reactions studied were the reduction of p‐nitro phenol and the reduction of hexacyanoferrate (III) by borohydride ions. Both the reactions were monitored by UV‐Vis spectroscopy. The kinetic investigations were carried out for the AuNPs‐catalyzed reactions at different temperatures and different amount of catalyst.     

Topochemical synthesis and catalysis of metal nanoparticles exposed on crystalline cellulose nanofibers

Topochemical synthesis of gold nanoparticles (AuNPs) was achieved on crystalline cellulose single nanofibers (CSNFs), which were tailored from native cellulose. Exposed AuNPs@CSNFs composite exhibited an excellent catalytic efficiency: the turnover frequency of the AuNPs@CSNFs was up to 840 times that of conventional polymer-supported AuNPs, for a model aqueous reduction reaction. Our novel strategy provides a promising solution to realize efficient use of limited noble metals using natural bioresources.     

Nonionic surfactant‐capped gold nanoparticles for selective enrichment of aminothiols prior to CE with UV absorption detection

In this study, we describe the use of Tween 20‐capped gold nanoparticles (AuNPs) as selective probes for the extraction of aminothiols from an aqueous solution. Tween 20 molecules noncovalently attached to the surface of AuNPs to form Tween 20–AuNPs were used for the selective extraction of aminothiols through the formation of Au–S bonds. After extraction and centrifugation, the aminothiols were detached from the surface of the AuNPs by adding DTT in a high concentration. We used this probe in combination with CE and UV absorption detection. On‐line concentration and separation of the released aminothiols were performed by using 1.6% v/v poly(diallyldimethylammonium chloride) as an additive in CE. Under optimal extraction and stacking conditions, the LOD at a S/N of 3 were 28, 554, and 456 nM for glutathione (GSH), cysteine (Cys), and homocysteine (HCys), respectively. In comparison with the normal injection without the extraction procedure, approximately 2280‐, 998‐, and 904‐fold improvements in the sensitivity were observed for GSH, Cys, and HCys, respectively. We have validated the application of our method on the basis of the analysis of GSH and HCys in human urine samples. It is believed that this approach has significant potential to be extended to clinical diagnosis.     

Small Size Effect and Concentration Response of Gold Nanoparticles in Electrochemiluminescence Reaction

Recent reports have used gold nanoparticles (AuNPs) as a co‐reactant for the electrochemiluminescence (ECL) reaction of ruthenium complex. However, understanding the size effect of AuNPs on ECL reaction is very meaningful to explore its unknowns and develop its applications at the molecular level. In this paper, we examined the behavior of various small‐size AuNPs in ECL reaction, focusing on changes in ECL caused by AuNPs size and reasons for this change. Although the luminescence spectra and excitation potential have hardly changed in ECL reaction, the difference of ECL intensities induced by different sizes AuNPs is very obviously. Our experimental results revealed disparate behaviors depending on AuNPs size: the small‐sized AuNPs can lead to stronger ECL, and ECL intensities increase as the addition of AuNPs concentration in the wider range. This small size effect is related to an intermediate process of charge‐discharge in electric double layer formed by adsorbing ruthenium complex with AuNPs, and the surface and quantum size effect of AuNPs may affect this intermediate process. More importantly, AuNPs can act as a marker, has the same small size effect and concentration response, and bring about a promising platform for biochemical analysis.