Polycatalytic enzyme complexes made by immobilization of industrial enzymes on polymer- or nanoparticle-based scaffolds are technologically attractive due to their recyclability and their improved substrate binding and catalytic activities. Herein, we report the synthesis of polycatalytic complexes by the immobilization of nonprocessive cellulases on the surface of colloidal polymers with a magnetic nanoparticle core and the study of their binding and catalytic activities. These polycatalytic cellulase complexes have increased binding affinity for the substrate. But due to their larger size, these complexes were unable to access to the internal surfaces of cellulose and have significantly lower binding capacity when compared to those of the corresponding free enzymes. Analysis of released soluble sugars indicated that the formation of complexes may promote the prospect of having consistent, multiple attacks on cellulose substrate. Once bound to the substrate, polycatalytic complexes tend to remain on the surface with very limited mobility due to their strong, multivalent binding to cellulose. Hence, the overall performance of polycatalytic complexes is limited by its substrate accessibility as well as mobility on the substrate surface.

For the highly efficient extraction of the N-glycoproteome, a novel solid-phase extraction method based on oxime click chemistry has been developed. With the use of a newly synthesized aminooxy-functionalized magnetic nanoparticle, the oxidized glycan chains on glycopeptides readily react with the aminooxy groups through oxime click chemistry, resulting in the highly selective extraction of glycopeptides. Compared to the traditional hydrazide chemistry-based method, which takes 12-16 h of coupling time, this new method renders excellent enrichment performance within 1 h. Furthermore, the enrichment sensitivity (fmol level), selectivity (extracting glycopeptides from mixtures of nonglycopeptides at a 1:100 molar ratio), and reproducibility (CVs < 20%) are also dramatically improved. We have successfully profiled the N-glycoproteome from only 1 μL of human colorectal cancer serum using this innovative protocol, which offers a more efficient alternative N-glycoproteome extraction method.

The efficient isolation of low-abundance phosphopeptides from complicated biological samples containing a significant quantity of nonphosphopeptides and proteins is essential for phosphopeptidome research but remains a great challenge. In this Article, magnetic composite microspheres comprising a magnetic colloidal nanocrystal cluster core and a mesoporous titania shell with an average pore diameter of 3.4 nm were modified by directly coating an amorphous titania shell onto the magnetite core, followed by converting the amorphous titania shell into a crystalline structure via a hydrothermal process at 80 °C. The as-prepared magnetic mesoporous titania microspheres possess a remarkable specific surface area that is as high as 603.5 m2/g, which is an appropriate pore size with a narrow size distribution and a high magnetic responsiveness. These outstanding features imply that the composite microspheres exhibit extraordinary performance in phosphopeptidome research, including high specificity toward phosphopeptides, an excellent size-exclusion effect against phosphoproteins, exceptional enrichment capacity, and efficient separation from mixtures. Encouraged by the experimental results, we employed this method to investigate the phosphopeptidome of snake venom for the first time. A total of 35 phosphopeptides was identified from the snake venom from the family Viperidae, accounting for 75% of the total identified peptides. This result represents the largest data set of the phosphopeptidome in snake venom from the family Viperidae.

The selective extraction of phosphopeptidome from complicated biological samples is of great importance for the development of diagnostic and prognostic biomarkers, but still remains a challenge. In this work, rattle-type mTiO(2)@P(NIPAM-co-MBA) composite microspheres comprising a mesoporous crystalline mTiO(2) core, an intermediate hollow space and a crosslinked P(NIPAM-co-MBA) network shell were elaborately designed and fabricated via two-step reflux-precipitation polymerization followed by a hydrothermal process. First, a non-crosslinked PMAA layer was directly coated onto the surface of the TiO2 core without any pretreatment. Then, the formed TiO2@PMAA was encapsulated with another crosslinked P(NIPAM-co-MBA) layer with the aid of the strong hydrogen-bonding interaction between the two polymer layers. Finally, a hydrothermal process was adopted to convert the TiO2 core into a crystalline and mesoporous counterpart. At the same time, a non-crosslinked PMAA layer was selectively removed to form a rattle-type structure. The crosslinked P(NIPAM-co-MBA) shell makes the rattle-type mTiO(2)@P(NIPAM-co-MBA) possess great size-exclusion effect against both high-molecular-weight nonphosphoproteins and high-molecular-weight phosphoproteins ,while the mTiO(2) core was responsible of the selective enrichment of the low-molecular-weight phosphopeptides. With the help of these unique properties, the rattle-type mTiO(2)@P(NIPAM-co-MBA) microspheres show excellent potential for the one-step selective extraction of the phosphopeptidome.

Efficient enrichment of specific glycoproteins from complex biological samples is of great importance towards the discovery of disease biomarkers in biological systems. Recently, phenylboronic acid-based functional materials have been widely used for enrichment of glycoproteins. However, such enrichment was mainly carried out under alkaline conditions, which is different to the status of glycoproteins in neutral physiological conditions and may cause some unpredictable degradation. In this study, on-demand neutral enrichment of glycoproteins from crude biological samples is accomplished by utilizing the reversible interaction between the cis-diols of glycoproteins and benzoboroxole-functionalized magnetic composite microspheres (Fe3O4/PAA-AOPB). The Fe3O4/PAA-AOPB composite microspheres are deliberately designed and constructed with a high-magnetic-response magnetic supraparticle (MSP) core and a crosslinked poly(acrylic acid) (PAA) shell anchoring abundant benzoboroxole functional groups on the surface. These nanocomposites possessed many merits, such as large enrichment capacity (93.9 mg/g, protein/beads), low non-specific adsorption, quick enrichment process (10 min) and magnetic separation speed (20 s), and high recovery efficiency. Furthermore, the as-prepared Fe3O4/PAA-AOPB microspheres display high selectivity to glycoproteins even in the E. coli lysate or fetal bovine serum, showing great potential in the identify of low-abundance glycoproteins as biomarkers in real complex biological systems for clinical diagnoses.

Integration of the advantages of immobilized metal-ion affinity chromatography (IMAC) and magnetic microspheres is considered as an ideal pathway for quick and convenient separation of his-tagged proteins, but rare reports concern the natural histidine-rich proteins. In this article, a novel route was presented to fabricate magnetic microspheres composed of a high-magnetic-response magnetic supraparticle (Fe3O4) core and a Ni(2+)-immobilized cross-linked polyvinyl imidazole (PVIM) shell via reflux-precipitation polymerization. The unique as-prepared Fe3O4/PVIM-Ni(2+) microspheres possessed uniform flower-like structure, high magnetic responsiveness, abundant binding sites, and very easy synthesis process. Taking advantage of the pure PVIM-Ni(2+) interface and high Ni(2+) loading amount, the microspheres exhibited remarkable selectivity, excellent sensitivity, large enrichment capacity, and high recyclability in immobilization and separation of his-tagged recombinant proteins. More interestingly, it was found that the Fe3O4/PVIM-Ni(2+) microspheres also showed excellent performance for removal of the natural histidine-rich bovine serum albumin (BSA) from the complex real sample of fetal bovine serum due to the exposed histidine residues. Considering their multiple merits, this new type of Fe3O4/PVIM-Ni(2+) nanomaterial displays great potential in enriching low-abundant his-tagged proteins or removing high-abundant histidine-rich natural proteins for proteomic analysis.

Magnetic yolk-shell MSP@ZrO2 microspheres consisting of a movable magnetic supraparticle (MSP) core and a crystalline ZrO2 shell were synthesized via a two-step controlled "sol-gel" approach for the first time. First, a large amount of the generated hydrolyzate Zr(OH)4 was firmly fixed onto the surface of the cross-linked polymethylacrylic acid matrix via a strong hydrogen-bonding interaction between Zr(OH)4 and the carboxyl groups. Then a calcination process was adopted to convert the Zr(OH)4 into a continuous ZrO2 shell and simultaneously make the ZrO2 shell crystallized. At the same time, the polymer matrix could be selectively removed to form a yolk-shell structure, which has better dispersibility and higher adsorbing efficiency of phosphopeptides than its solid counterpart. The formation mechanism of such yolk-shell microspheres could be reasonably proved by the results of TEM, TGA, VSM, XRD, and FT-IR characterization. By taking advantage of the unique properties, the yolk-shell MSP@ZrO2 exhibited high specificity and great capability in selective enrichment of phosphopeptides, and a total of 33 unique phosphopeptides mapped to 33 different phosphoproteins had been identified from 1 mL of human saliva. This result clearly demonstrated that the yolk-shell MSP@ZrO2 has great performance in purifying and identifying the low-abundant phosphopeptides from real complex biological samples. Moreover, the synthetic method can be used to produce hybrid yolk-shell MSP@ZrO2-TiO2.

An ultrafast, green and efficient microwave-assisted hydrothermal crystallization method was developed to convert the amorphous titania shell of MSP@TiO2 to uniform mesoporous anatase structure for highly selective and effective enrichment of phosphopeptides.

Designed with a two-in-one strategy, the magnetic mesoporous γ-Fe(2)O(3) nanocrystal clusters (m-γ-Fe(2)O(3)) have been successfully prepared for integrating the functions of effective enrichment and quick separation of phosphopeptides into a single architecture. First, the mesoporous Fe(3)O(4) nanocrystal clusters (mFe(3)O(4)) were synthesized by solvothermal reaction and then were subjected to calcination in air to form m-γ-Fe(2)O(3). The obtained m-γ-Fe(2)O(3) have spherical morphology with uniform particle size of about 200 nm and mesoporous structure with the pore diameter of about 9.7 nm; the surface area is as large as 117.8 m(2)/g, and the pore volume is 0.34 cm(3)/g. The m-γ-Fe(2)O(3) possessed very high magnetic responsiveness (Ms = 78.8 emu/g, magnetic separation time from solution is less than 5 s) and were used for the selective enrichment of phosphopeptides for the first time. The experimental results demonstrated that the m-γ-Fe(2)O(3) possessed high selectivity for phosphopeptides at a low molar ratio of phosphopeptides/nonphosphopeptides (1:100), high sensitivity (the detection limit was at the fmol level), high enrichment recovery (as high as 89.4%), and excellent speed (the enrichment can be completed in 10 min). Moreover, this material is also quite effective for enrichment of phosphopeptides from the real sample (drinking milk), showing great potential in the practical application.

The formation of polycatalytic enzyme complexes may enhance the effectiveness of enzymes due to improved substrate interaction and synergistic actions of multiple enzymes in proximity. Much effort has been made to develop highly efficient polycatalytic cellulase complexes by immobilizing cellulases on low-cost polymer or nanoparticle scaffolds, aiming at their potential applications in biomass conversion to fuels. However, some key cellulases carry out the hydrolytic reaction on crystalline cellulose in a directional, processive manner. A large, artificial polycatalytic complex is unlikely to undergo a highly coordinated motion to slide on the cellulose surface as a whole unit. The mechanism underlying the activity enhancements observed in some artificial cellulase complexes and the limit of this approach remain elusive. Herein, we report the synthesis of polycatalytic cellulase complexes bound to colloidal polymer nanoparticles with a magnetic core and describe their unique adsorption, hydrolytic activities, and motions on cellulose. The polycatalytic clusters of cellulases on colloidal polymers show an increased rate of hydrolytic reactions on cellulose, but this was observed mainly at relatively low cellulase-to-cellulose ratios. Enhanced efficiency is mainly attributed to increased local concentrations of cellulases on the scaffolds and their polyvalent interactions with cellulose. However, once bound, the polycatalytic complexes can only carry out reactions locally and are not capable of relocating to new sites rapidly due to their lack of long-range surface mobility and their extremely tight binding. The development of highly optimized polycatalytic complexes may arise by developing novel nanoscaffolds that induce concerted motion of the complex as a whole.

A new approach for sensitive detection of a specific ssDNA (single-stranded DNA) sequence based on the surface enhanced Raman spectroscopy (SERS) liquid chip is demonstrated. In this method, the probe DNA (targeting to one part of target ssDNA) was attached to the nano-SERS-tags (poly(styrene-co-acrylic acid)/(silver nanoparticles)/silica composite nanospheres), and the capture DNA (targeting to the other part of target ssDNA) was attached to the Fe3O4/poly(acrylic acid) core/shell nanospheres. The nano-SERS-tags with probe DNA were first allowed to undergo hybridization with the target ssDNA in solution to achieve the best efficiency. Subsequently, the magnetic composite nanospheres with capture DNA were added as the capturing substrates of the target ssDNA combined with the nano-SERS-tags. Upon attraction with an external magnet, the nanospheres (including the nano-SERS-tags) were deposited together due to the hybridization, and the deposit sediment was then analyzed by SERS. Quantitative detection of target ssDNA was achieved based on the well-defined linear correlation between the SERS signal intensity and the target ssDNA quantity in the range of 10 nM to 10 pM, and the limit of detection was approximately 10 pM. Multiplexed detection of up to three different ssDNA targets in one sample was demonstrated using three different types of nano-SERS-tags under a single excitation laser. The experimental results indicated that the liquid-phase DNA sequencing method, thus named the SERS liquid chip (SLC) method, holds significant promises for specific detection of trace targets of organisms.

A facile approach has been developed to synthesize Fe3O4/PMG (poly (N,N'-methylenebisacrylamide-co-glycidyl methacrylate)) core/shell microspheres using distillation-precipitation polymerization. Treating PMG shell with iminodiacetic acid (IDA) and Ni2+ yields composite microspheres of Fe3O4/PMG/IDA-Ni2+. The Ni2+ ions loaded on the surface of microspheres provide abundant docking sites for immobilization of histidine-tagged proteins. The high saturation magnetization of Fe3O4/PMG (23 emu/g), determined by vibrating sample magnetometer (VSM), allows an easy separation of the microspheres from solution under an external magnetic field. The composite microspheres were used to purify two His-tagged cellulolytic enzymes (Cel48F and Cel9G) directly from crude cell lysates with high binding affinity, capacity, and specificity. The microspheres can be recycled for many times without significant loss of binding capacity to enzymes. The immobilized enzymes on the surface of microspheres well retain their biological activities in degradation of cellulose. These materials show great potential in the biomedical and biotechnological applications that require low-cost purification of recombinant proteins and instant enzyme immobilization at an industrial scale.

An ultrafast, facile, and efficient microwave hydrothermal approach was designed to fabricate magnetic Fe(3)O(4)/phenol-formaldehyde (PF) core-shell microspheres for the first time. The structure of the Fe(3)O(4)/PF core-shell microspheres could be well controlled by the in situ polycondensation of phenol and formaldehyde with magnetic Fe(3)O(4) clusters as the seeds in an aqueous solution without any surfactants. The effect of synthetic parameters, such as the feeding amounts of phenol, the dosages of formaldehyde, the reaction temperatures, and the microwave heating time, on the morphologies and sizes of the Fe(3)O(4)/PF microspheres were investigated in details. The phenol-formaldehyde shell is found to be evenly coated on Fe(3)O(4) clusters within 10 min of the irradiation. The as-prepared microspheres were highly uniform in morphology, and the method was found to allow the shell thickness to be finely controlled in the range of 10-200 nm. The properties of the composite microspheres were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), thermogravimetic analysis (TGA), Fourier transform infrared (FT-IR) spectra, X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). The as-prepared Fe(3)O(4)/PF microspheres were monodisperse and highly dispersible in water, ethanol, N,N-dimethyformamide, and acetone, a beneficial quality for the further functionalization and applications of the Fe(3)O(4)/PF microspheres.

Well-defined amphiphilic diblock copolymers of poly(N-(2-hydroxypropyl)methacrylamide)-block-poly(benzyl methacrylate) (PHPMA-b-PBnMA) are synthesized using reversible additionfragmentation chain transfer polymerization. The terminal dithiobenzoate groups are converted into carboxylic acids. The copolymers self-assemble into micelles with a PBnMA core and PHPMA shell. Their mean size is <30 nm, and can be regulated by the length of the hydrophilic chain. The compatibility between the hydrophobic segment and the drug doxorubicin (DOX) affords more interaction of the cores with DOX. Fluorescence spectra are used to determine the critical micelle concentration of the folate-conjugated amphiphilic block copolymer. Dynamic light scattering measurements reveal the stability of the micelles with or without DOX. Drug release experiments show that the DOX-loaded micelles are stable under simulated circulation conditions and the DOX can be quickly released under acidic endosome pH.

Selective enrichment of glycopeptides from complicated biological samples is essential for MS-based glycoproteomics, but still remains a great challenge. In this study, we report an unprecedented ligand-free strategy for the selective enrichment of glycopeptides by simply utilizing the multivalent interaction between glycopeptides and silver nanoparticles (Ag-NPs) coated magnetic nanoarchitectures. The composite microspheres were deliberately designed to be constructed with a high-magnetic-response magnetic colloid nanocrystal cluster (MCNC) core, a poly(methacrylic acid) (PMAA) interim layer and a Ag-NPs functional shell with high coverage. Taking advantage of the reversible interaction of glycans with Ag-NPs and the high magnetic susceptibility of the magnetite core, the MCNC@PMAA@Ag-NPs microspheres possess remarkable selectivity for glycopeptides even at a low molar ratio of glycopeptides/non-glycopeptides (1 : 100) with a very rapid enrichment speed (only 1 min needed) and a simple operation procedure using magnetic separation. Applying this approach, we identified 127 unique glycopeptides mapped to 51 different glycoproteins from only 1 μL rat serum samples. These results clearly demonstrated that the MCNC@PMAA@Ag-NPs have great potential for purifying and identifying the low-abundant glycopeptides in complex biological samples.

The heterostructure Ag-Au bimetallic nanocrystals supported on Fe3O4@carbon composite microspheres were synthesized by one facile and controllable approach, wherein the Ag nanocrystals attached on the Fe3O4@carbon microspheres were prepared first and served as reductant for the galvanic replacement reaction with the Au precursor (HAuCl4). Upon varying the feeding amounts of the Au precursor, the bimetallic compositions on the Fe3O4@carbon microsphere could be readily tuned resulting in a series of composite microspheres with different Au-to-Ag molar ratios. Subsequently, we thus investigated the catalytic activity and selectivity of the magnetic composite catalysts from two sides. First, 4-nitrophenol (4-NP) was applied as a model molecule to study the effect of different Au-to-Ag molar ratios on catalytic capabilities of the resulting composite microspheres. It was found that upon the addition of NaBH4 the catalytic capability was markedly enhanced when the Au content was increased. The maximum activity parameter value reached 1580 which is far higher than those of known monometallic composites. Also, they could give the equally high yields for other nitroaromatic compounds with various substituents, irrespective of the linked electron donating or electron withdrawing groups. Second, the synergistic effects of the carbon substrate in the catalysis reaction were demonstrated. When compared with colloidal SiO2, TiO2, and poly(styrene-co-acrylic acid) substrates, the carbon support not only facilitated the enhancement of the catalytic performance of the noble metal nanocrystals but was also more suitable for the in situ preparation of Au Ag bimetallic nanocrystals using the GRR. Besides, the particles' convenience in terms of their magnetic separability and outstanding reusability was validated through many successive reduction reaction cycles. In light of these unique characteristics, the Fe3O4@C@Ag-Au composite microspheres show promising and great potential for practical applications.

An effective method was developed for synthesizing magnetite/polymer colloidal composite microspheres with controllable variations in size and shape of the nanostructures and desirable interfacial chemical functionalities, using surfactant-free seeded emulsion polymerization with magnetite (Fe(3)O(4)) colloidal nanocrystal clusters (CNCs) as the seed, styrene (St) as the monomer, and potassium persulfate (KPS) as the initiator. The sub-micrometer-sized citrate-acid-stabilized Fe(3)O(4) CNCs were first obtained via ethylene glycol (EG)-mediated solvothermal synthesis, followed by 3-(trimethoxysilyl)propyl methacrylate (MPS) modification to immobilize the active vinyl groups onto the surfaces, and then the hydrophobic St monomers were polymerized at the interfaces to form the polymer shells by seeded emulsion radical polymerization. The morphology of the composite microspheres could be controlled from raspberry- and flower-like shapes, to eccentric structures by simply adjusting the feeding weight ratio of the seed to the monomer (Fe(3)O(4)/St) and varying the amount of cross-linker divinyl benzene (DVB). The morphological transition was rationalized by considering the viscosity of monomer-swollen polymer matrix and interfacial tension between the seeds and polymer matrix. Functional groups, such as carboxyl, hydroxyl, and epoxy, can be facilely introduced onto the composite microspheres through copolymerization of St with other functional monomers. The resultant microspheres displayed a high saturation magnetization (46 emu/g), well-defined core-shell nanostructures, and surface chemical functionalities, as well as a sustained colloidal stability, promising for further biomedical applications.

Selective enrichment of phosphoproteins or phosphopeptides from complex mixtures is essential for MS-based phosphoproteomics, but still remains a challenge. In this article, we described an unprecedented approach to synthesize magnetic mesoporous Fe(3)O(4)@mTiO(2) microspheres with a well-defined core/shell structure, a pure and highly crystalline TiO(2) layer, high specific surface area (167.1 m(2)/g), large pore volume (0.45 cm(3)/g), appropriate and tunable pore size (8.6-16.4 nm), and high magnetic susceptibility. We investigated the applicability of Fe(3)O(4)@mTiO(2) microspheres in a study of the selective enrichment of phosphopeptides. The experiment results demonstrated that the Fe(3)O(4)@mTiO(2) possessed remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/non-phosphopeptides (1:1000), large enrichment capacity (as high as 225 mg/g, over 10 times as that of the Fe(3)O(4)@TiO(2) microspheres), extreme sensitivity (the detection limit was at the fmol level), excellent speed (the enrichment can be completed in less than 5 min), and high recovery of phosphopeptides (as high as 93%). In addition, the high magnetic susceptibility allowed convenient separation of the target peptides by magnetic separation. These outstanding features give the Fe(3)O(4)@mTiO(2) composite microspheres high benefit for mass spectrometric analysis of phosphopeptides.

A smart magnetic targeting drug carrier (MCNC/PAA) comprising an approximately 100 nm sized magnetic colloid nanocrystal cluster (MCNC) core and a pH-responsive cross-linked poly(acrylic acid) (PAA) shell is reported. The abundant carboxyl groups in the shell enable the resultant MCNC/PAA to easily load a large amount of doxorubicin (DOX) (up to 44.6%) via the strong interaction between the DOX and the carboxyl group in a neutral solution. Interestingly, a synergistic pH-responsive effect derived from the entrapped DOX and PAA network was found to effectively manipulate the drug releasing behavior at 37 °C. It was found that the premature release was highly restricted at a pH of 7.4, and upon reduction in pH from 7.4 to 5.0 or 4.0, a large amount of drug was rapidly released. Compared with the synthesized MCNC/PNIPAM, MCNC/PHEMA and MCNC/PDMAPMA nanocarriers, the MCNC/PAA was preferably suited to drug delivery. In addition, the composite nanocarriers could be tracked by magnetic resonance imaging (MRI). The cytotoxicity assay of MCNC/PAA to normal cells indicated that the composite nanospheres were biocompatible and suitable as drug carriers. Meanwhile, the DOX-loaded composite nanospheres had more potent cytotoxicity than free DOX to HeLa cells. These results clearly imply that the MCNC/PAA nanocarrier is a promising platform that can be applied to construct a smart drug delivery system with magnetic targeting and pH-stimulation, as well as tracking by MRI.

Architectural design is essential to achieve ideal chemical and biological properties of nanomaterials. In this article, a novel route to fabricate high‐quality magnetic composite microspheres composed of a high‐magnetic‐response magnetic colloid nanocrystal cluster (MCNC) core, a poly(methylacrylic acid) (PMAA) interim layer, and a Ti4+‐immobilized poly(ethylene glycol methacrylate phosphate) (PEGMP) shell via two‐step distillation–precipitation polymerization is presented. The unique as‐synthesized MCNC@PMAA@PEGMP‐Ti4+ composite microsphere is investigated for its applicability for selective enrichment of phosphopeptides from complex biological samples. The experiment results demonstrate that, by taking advantage of the pure phosphate–Ti4+ interface and high Ti4+ loading amount, the MCNC@PMAA@PEGMP‐Ti4+ composite microsphere possesses remarkable selectivity for phosphopeptides even at a very low molar ratio of phosphopeptides/nonphosphopeptides (1:500). The extreme sensitivity, excellent recovery of phosphopeptides, and high magnetic susceptibility are also proven. These outstanding features demonstrate that the MCNC@PMAA@PEGMP‐Ti4+ composite microspheres have great benefit for the pretreatment before mass spectrometric analysis of phosphopeptides. Furthermore, the performance of the approach in selective enrichment of phosphopeptides from drinking milk and human serum gives powerful evidence for its high selectivity and effectiveness in identifying the low‐abundant phosphopeptides from complicated biological samples.