Amino-functionalized Silica Nanoparticles Research Articles

Synthesis and Characterization of Hyaluronic Acid Modified Colloidal Mesoporous Silica Nanoparticles

The colloidal mesoporous silica nanoparticles functionalized with hyaluronic acid (CMS-HA) were successfully synthesized by grafting hyaluronic acid onto the external surface of the amino-functionalized mesoporous silica nanoparticles (CMS-NH2). Moreover, the paticle properties of CMS-HA were characterized by fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and transmission electron microscopy (TEM). The nanomaterials were negatively charged and had a relatively uniform spherical morphology with about 100 nm in diameter, which could make it more compatible with blood. So the results suggested that the CMS-HA might be a critical nanomaterial for applying in target drug delivery system.

Nanoparticle core stability and surface functionalization drive the mTOR signaling pathway in hepatocellular cell lines

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization and biodegradability. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH2), but not amino- or hydroxyl-functionalized silica particles, trigger cell death in hepatocellular carcinoma Huh7 cells. Importantly, biodegradability of nanoparticles plays a crucial role in regulation of essential cellular processes. Thus, biodegradable silica nanoparticles having the same shape, size and surface functionalization showed opposite cellular effects in comparison with similar polystyrene nanoparticles. At the molecular level, PS-NH2 obstruct and amino-functionalized silica nanoparticles (Si-NH2) activate the mTOR signalling in Huh7 and HepG2 cells. PS-NH2 induced time-dependent lysosomal destabilization associated with damage of the mitochondrial membrane. Solely in PS-NH2-treated cells, permeabilization of lysosomes preceded cell death. Contrary, Si-NH2 nanoparticles enhanced proliferation of HuH7 and HepG2 cells. Our findings demonstrate complex cellular responses to functionalized nanoparticles and suggest that nanoparticles can be used to control activation of mTOR signaling with subsequent influence on proliferation and viability of HuH7 cells. The data provide fundamental knowledge which could help in developing safe and efficient nano-therapeutics.

Synthesis of amino-functionalized silica nanoparticles for preparation of new laboratory standards

Platinum group elements (PGEs) are particularly interesting analytes in different fields, including environmental samples as well as high cost materials that contain them, such as for example automotive catalysts. This type of solid samples could be analysed by laser ablation (LA) coupled to ICP-MS, which allow to significantly reducing the analysis time since the time-consuming processes for sample preparation are not required. There is a considerable demand of standards with high PGEs concentration for quantification purposes, which cannot be carried out easily using LA-ICP-MS because the available standards (i.e. NIST SRM 61× series) do not have such analytes in the same concentration range. In this paper, a new strategy is proposed for the synthesis of homogeneous laboratory standards with Pt, Pd and Rh concentrations that range from 77μg/g of Pd up to 2035μg/g of Rh. The proposed strategy is based on the synthesis of monodisperse amino-functionalized amorphous silica nanoparticles, which can retain metal ions. In addition to Pt, Pd and Rh, three lanthanides were also added to the nanoparticles (La, Ce, Nd). Sturdy pressed pellets can be made from the resulting nanopowder without the use of any binder. Elemental composition of standards made of nanoparticles was analysed by conventional nebulization ICP-MS and their homogeneity was successfully evaluated by LA-ICP-MS.

Dendrimer-like amino-functionalized hierarchical porous silica nanoparticle: A host material for 2,4-dichlorophenoxyacetic acid imprinting and sensing

In this work, a novel molecularly imprinted electrochemical sensor based on the amino-functionalized silica nanoparticles was built for the sensitive and selective detection of 2,4-dichlorophenoxyacetic acid (2,4-D). The hierarchical porous dendrimer-like silica nanoparticles (HPSNs-NH2) were synthesized by an ethyl ether emulsion method. The selective molecularly imprinted polymers (MIP) was prepared on the HPSNs-NH2 modified electrode via electropolymerization by using 2,4-D as the template and o-phenylenediamine (OPD) as the monomer. The porous structure of HPSNs-NH2 reduced the diffusion limitations of the analytes, enhanced the accessibility and increased the surface area of the sensor, while the MIP layer offered the ability to recognize and quantify target 2,4-D by using ferro/ferricyanide as probes. Several significant experimental parameters on the analytical performance of the MIP/HPSNs-NH2 sensor were explored and optimized. Under the optimized condition, the sensor displayed an appreciable selectivity over structurally related compounds and good sensitivity toward 2,4-D. The linear range of 2,4-D detection was from 1.00 × 10−10 to 2.50 × 10−8M and the detection limit was down to 1.17 × 10–11M according to the 3Sa/b criteria. This method has been applied to detect 2,4-D in bean sprout samples with satisfying results.

Amino-functionalized mesoporous silica nanoparticles as efficient carriers for anticancer drug delivery.

Amino-functionalized mesoporous silica nanoparticles (MSN-NH2) were synthesized by a post-grafting method and further studied as carriers for doxorubicin hydrochloride (DOX) delivery. The morphology, structure, and property of MSN-NH2 and DOX-loaded MSN-NH2 (DOX@MSN-NH2) were studied using various techniques, such as transmission electron microscopy, Fourier transformed infrared spectroscopy, N2 adsorption-desorption isotherms, and zeta potentials. The drug loading and release profile as well as the invitro cell cytotoxicity were detaily investigated. The results indicated that the loading content of DOX increased with the decrease of MSN-NH2/DOX mass ratio and/or the increase of amino density. DOX@MSN-NH2 exhibited a pH-dependent drug release, drug release increased as the pH value decreased. Compared with MSN-NH2, which were neglectable cytotoxicity against non-small-cell lung cancer (A549) cells, DOX@MSN-NH2 displayed remarkable cytotoxicity toward A549 cells in dose- and time-dependent manners. It was concluded that the as-synthesized MSN-NH2 could be used as promising drug carriers for cancer therapy.

Large photoacoustic effect enhancement for ICG confined inside MCM-41 mesoporous silica nanoparticles.

Indocyanine green was encapsulated inside the pores of pegylated amino-functionalized MCM-41 Mesoporous Silica Nanoparticles (ICG-MSNs). In addition to a greater stability and a decrease of toxicity, the photoacoustic effect of ICG-MSNs increases by nearly 400% compared to free ICG due to fluorescence quenching and high photothermal conversion of the encapsulated dyes. Upon i.v. administration in tumor-bearing mice, an overall photoacoustic enhancement of ca. 25% was measured in the tumor region.

Tailored Silica Nanoparticles Surface to Increase Drug Load and Enhance Bactericidal Response

Nanoparticles' surface properties can be used as triggers to regulate or even enhance biological response and generate tailored structures to substitute conventional antibiotics. Here, silica nanoparticles surface was duly tuned in order to increase the water-insoluble drug load (curcumin) and improve the antibacterial activity. Our main motivation was based on the electrostatic attraction between the positively charged amino groups and the negatively charged curcumin and/or bacteria membrane. In addition, the variation of amino grafting amount on silica nanoparticles indicated that the grafting increase was directly related to the extent of drug entrapped into the nanoparticles as well as to the bactericidal activity. The combination of amino-functionalized silica nanoparticles associated with the presence of curcumin allowed to produce a dual bactericidal system that shows promising perspective for its use in biomedical applications.

Induction of Superhydrophobicity in a Cellulose Substrate by LbL Assembly of Covalently Linked Dual-Sized Silica Nanoparticles Layers

Micro/nano texturized oxidized cellulose membranes (MNOCM) were constructed by layer-by-layer (LbL) assembly in which a base cellulose film was modified by covalent linkages to amino-functionalized silica nanoparticles (amino-SiO2 NPs, 260 nm diameter) and epoxy-functionalized silica nanoparticles (epoxy-SiO2 NPs, 30 nm diameter). The amino-SiO2 NPs grafted onto the MNOCM surface through a standard amidation reaction between the amino groups of the SiO2 NPs and the carboxyl groups of the MNOCM surface in the presence of EDC and NHS consequently forming a first layer of large (260 nm) nanoparticles; subsequently, it was reacted with smaller (30 nm) epoxy-SiO2 NPs. Continuous repetitions of these alternating sized silica NPs through a standard LbL approach lead to a highly micro/nano-texturized MNOCM film as shown by SEM, which was ultimately sealed with a layer of hydrophobic PFOTES (1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane). Although the wettability of MNOCM was no longer hydrophilic, it was found that at five layers deep of NPs, it became superhydrophobic as evidenced by a water contact angle of 151° ± 2° and slide angle of 4°. The change in wettability was attributed to increases in final LbL layer surface roughness induced by the sufficient LbL layering of alternating sizes of NPs akin to what is observed in a lotus leaf surface. It was also noted that these superhydrophobic-MNOCM materials displayed good self-cleaning.

Fluorescence Anisotropy as a Reliable Discrimination of Ligand-Asymmetric and Symmetric Mn-Doped ZnS Quantum Dots.

We presented a novel fluorescence anisotropy (FA) method for the noninvasive, effective, simple, and convenient discrimination of the symmetric and asymmetric distribution of the ligands on Mn-doped ZnS quantum dots (QDs). The symmetric or asymmetric distribution of mercaptopropionic acid (MPA) and NH2-polyethylene glycol-CH3 (PEG-m, MW 2000) was controlled by the condensation reaction of the carboxyl of MPA and the amino of PEG-m with or without the masking by the amino-functionalized silica nanoparticles. The ligand-asymmetric Janus-QDs were obtained with the masking, whereas the ligand-symmetric PEG-QDs were gained without masking. The FA values of the QDs could not only distinguish the ligand symmetric PEG-QDs from the ligand asymmetric Janus-QDs but also discriminate the QDs with a different PEG-m amount. Besides, the FA assay also has superiority over the dynamic light scattering (DLS) and photoluminescence (PL) methods in discriminating the interaction of Janus-QDs or PEG-QDs with protamine (the sensitivity of Janus-QD-3 over PEG-QD-3 was 1.60, 1.24, and 1.11 in the FA, DLS, and PL methods, respectively).

PH-Responsive Core-Shell Structured Nanoparticles for Triple-Stage Targeted Delivery of Doxorubicin to Tumors.

The application of cytotoxic chemotherapeutics in cancer therapy has been largely restricted by their lack of selectivity. Despite the existence of numerous targeted delivery systems, it is practically challenging to develop one single system to simultaneously cover tumor-targeted delivery of chemotherapeutics at the tissue, cellular, and subcellular levels. To solve this problem, pH-responsive core-shell structured nanoparticles (CSNPs) were self-assembled in this study to provide triple-stage targeted delivery of doxorubicin (DOX) from the injection site to the nuclei of cancer cells. Amino-functionalized mesoporous silica nanoparticles (MSN) were doubly modified with TAT peptide and acid-cleavable polyethylene glycol (PEG) as the DOX-loaded cationic core. The anionic shell was constituted by galactose-modified poly(allylamine hydrochloride)-citraconic anhydride, a hepato-carcinoma-targeting polymer with charge-reversible property. In vitro results showed that PEG effectively reduced protein adsorption and phagocytic capture of CSNPs in the circulating blood (pH 7.4), thus facilitating passive accumulation in tumors (tissue level). Following PEG detachment via acidic hydrolysis in tumor microenvironment (pH 6.5), the exposed galactose ligands endowed CSNPs with active internalization into hepato-carcinoma cells (cellular level). Afterward, the acidity in endosomes and lysosomes (pH 5.0) triggered the conversion of anionic shell into positive charges, leading to core-shell disassembly and subsequent TAT-mediated delivery of DOX to the nuclei (subcellular level). Importantly, the efficiencies of each targeting moiety were nicely preserved when combining together in CSNPs. As a result, improved tumorous distribution and potent therapeutic efficacy of CSNPs were noted in tumor-bearing mice at a relatively low dose. CSNPs therefore provide an efficient and nontoxic platform for the targeted delivery of antitumor drugs.

Synthesis of a dodecylphenylsiloxane oligomer resin/silica nanocomposite and its application to cotton fabrics

A novel dodecylphenylsiloxane oligomer resin/nanocomposite (PHDESR-SiO2) was prepared by graft copolymerization between dodecyl modified phenylsiloxane resin with pendent epoxy groups (PHDESR) and amino-functionalized silica nanoparticles (BTEPA-SiO2). PHDESR-SiO2 was then used to prepare a super hydrophobic surface on cotton fabric by a facile solution-immersion process method. Chemical structures, chemical compositions, wettability, surface morphology, and thermal properties were investigated by Fourier Transform Infrared Spectrum (FT-IR), 1H-NMR spectrum, X-ray photoelectron spectroscopy (XPS), static contact angle analyzer, scanning electron microscopy (SEM), Particle size distribution (PSD) and thermo-gravimetric analysis (TGA). The results showed that the target product PHDESR-SiO2 has an anticipative structure with many micro/nanostructure tubercles, a cross-linked network hydrophobic organosilicon resin film and many clusters of cylindrical dodecyl molecular brushes. This created super hydrophobic structure on the surface of the treated cotton fabrics. XPS analysis indicated that the long carbon chain groups had a slight tendency to enrich the film-air interface. In addition, PHDESR-SiO2 can provide good hydrophobicity for the treated fabric. As the dose of PHDESR-SiO2 increased, the hydrophobicity of the treated fabric enhanced and consequently the water static contact angle reached 152.5 °. This had little influence on the softness, color, and gas permeability of the fabrics. This makes it slightly stiff at high doses, and the super-hydrophobic cotton fabric also had good launderability.

Rapid and sensitive detection of hemoglobin with gold nanoparticles based fluorescence sensor in aqueous solution

A novel gold nanoparticles (AuNPs)-based fluorescence assay had been developed for the detection of hemoglobin (Hb). The method relied on the high selectivity of surface imprinting technique and the strong fluorescence property of AuNPs. Herein, firstly, the carboxyl modified AuNPs were initially anchored on the amino-functionalized silica nanoparticles under the assistance of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxy-5-pyrrolidinedione (NHS). Then, the Hb-imprinted polymer layer was wrapped on the surface of SiO2/AuNPs via a sol–gel surface imprinting technique. The generated SiO2/Au/MIPs as fluorescence sensor owned both sensitive detection of AuNPs and specific selectivity of molecularly imprinted polymer. The conditions such as pH, stability and effect of the fluorescence sensor concentration were investigated to search the optimized detection conditions. For the optimized conditions, the fluorescence-quenching detection presented a satisfactory linearity with Hb concentrations in the range of 0.1–20 μmol L−1 and the detection limit was 0.03 μmol L−1. Moreover, through the investigation of selective and competitive quenching, the fluorescence sensor exhibited satisfying selectivity and high binding affinity to Hb under the existence of its possibly competing molecules, such as bovine serum albumin (BSA), bovine hemoglobin (BHb), albumin, chicken egg (ACE). The results proved that the fluorescence sensor was succeeded to be used in the field of rapid recognition and determination of Hb even though existing of other complex proteins.

Functionalized Silica Nanoparticles As an Alternative Platform for Targeted Drug-Delivery of Water Insoluble Drugs.

The selective action of drugs in tumor cells is a major problem in cancer therapy. Most chemotherapy drugs act nonspecifically and damage both cancer and healthy cells causing various side effects. In this study, the preparation of a selective drug delivery system, which is able to act as a carrier for hydrophobic and anticancer drugs is reported. Amino-functionalized silica nanoparticles loaded with curcumin were successfully synthesized via sol-gel approach and duly characterized. Thereafter, the targeting ligand, folate, was covalently attached to amino groups of nanoparticle surface through amide bond formation. The cytotoxic effect of nanoparticles on prostate cancer cells line was evaluated and compared to normal cells line (prostate epithelial cell). Cytotoxicity experiments demonstrated that folate-functionalized nanoparticles were significantly cytotoxic to tumor cells, whereas normal cells were much less affected by the presence of these structures.

Controlling the Interaction and Non-Close-Packed Arrangement of Nanoparticles on Large Areas.

In light of the importance of nanostructured surfaces for a variety of technological applications, the quest for simple and reliable preparation methods of ordered, nanometer ranged structures is ongoing. Herein, a versatile method to prepare ordered, non-close-packed arrangements of nanoparticles on centimeter sized surfaces by self-assembly is described using monodisperse (118-162 nm Ø), amino-functionalized silica nanoparticles as an exploratory example. It is shown that the arrangement of the particles is governed by the interplay between the electrostatic repulsion between the particles and the interaction between particles and surfaces. The latter is tuned by the properties of the particles such as their surface roughness as well as the chemistry of the linkage. Weak dispersive interactions between amino groups and gold surfaces are compared to a covalent amide linkage of the amino groups with carboxylic acid functionalized self-assembled monolayers. It was shown that the order of the former systems may suffer from capillary forces between particles during the drying process, while the covalently bonded systems do not. In turn, covalently bonded systems can be dried quickly, while the van der Waals bonded systems require a slow drying process to minimize aggregation. These highly ordered structures can be used as templates for the formation of a second, ordered, non-close-packed layer of nanoparticles exemplified for larger polystyrene particles (Ø 368 ± 14 nm), which highlights the prospect of this approach as a simple preparation method for ordered arrays of nanoparticles with tunable properties.

Co-delivery of erlotinib and doxorubicin by pH-sensitive charge conversion nanocarrier for synergistic therapy

Pretreatment of lung cancer cells with epidermal growth factor receptor (EGFR) inhibitor erlotinib has been recently reported that could dramatically synergize their apoptotic response to DNA damage agent doxorubicin (DOX). To translate this synergistic therapy into in vivo anticancer therapy and clinical practice, we designed a novel pH-sensitive charge conversion nanocarrier (M-HHG2C18-L) that contained erlotinib/DOX combination and produced a sequential staggered drug release for synergistic lung cancer therapy. In this study, a synthetic zwitterionic oligopeptide lipid (1,5-dioctadecyl-l-glutamyl2-histidyl-hexahydrobenzoic acid, HHG2C18) was used to construct a pH-sensitive lipid bilayer (HHG2C18-L), which was subsequently applied to coat amino-functionalized mesoporous silica nanoparticles (MSN-NH2). Erlotinib and DOX were separately incorporated into HHG2C18-L and MSN-NH2 respectively to obtain pH-sensitive charge conversion erlotinib/DOX co-delivery nanoparticles (M-HHG2C18-L(E+D)). We confirmed that M-HHG2C18-L(E+D) were able to reverse surface zeta potential from negative to positive at tumor extracellular pH, thus facilitating the targeted cancer cell internalization. Furthermore, as erlotinib was sequestered in the exterior lipid bilayer and the controlled release ability of MSN-NH2, erlotinib released faster than DOX during the cellular transport. Additionally, HHG2C18-L became more positive at tumor intracellular pH and enhanced Coulombic repulsion with MSN-NH2, leading to increased sequential staggered release of erlotinib and DOX. Due to the pretreatment and time-staggered inhibition of EGFR with erlotinib and the enhanced intracellular release of DOX to the nucleus, the maximized synergistic cell killing effect was achieved. Compared to non-sensitive erlotinib/DOX co-delivery nanoparticles (M-SPC-L(E+D)) and simultaneous DRUG coadministration. M-HHG2C18-L(E+D) with sequential staggered drug release and pH-sensitive charge conversional properties showed great synergistic effects in antiproliferation and apoptosis of A549 human cancer cells in vitro. The in vivo study demonstrated that M-HHG2C18-L(E+D) exhibited considerable tumor accumulation and potent suppression of tumor growth in Lewis lung carcinoma tumor bearing mice. It was also demonstrated that M-HHG2C18-L(E+D) showed no systemic toxicity and possessed distinguished effect on extending survival period. These results suggested that M-HHG2C18-L(E+D) had great potential application in cancer treatment.

One-pot synthesis and application of novel amino-functionalized silica nanoparticles using guanidine as amino group

The preparation of modified silica nanoparticles with guanidine was developed and used to catalyze the Henry reaction and fix quantum dots.

Control of particle uptake kinetics from particulate mesoporous silica films by cells through covalent linking of particles to the substrate - towards sequential drug delivery for tissue engineering applications.

A sequential release of biological cues is of high interest in tissue engineering applications, as both the proliferation and the differentiation of stem cells can be drugged. In previous studies we have shown that particulate films of mesoporous silica particles can be successfully applied for influencing stem cell fates through intracellular drug delivery after particle internalisation by the cells. In this study, we develop this concept towards an enhanced control of the particle uptake kinetics through either adsorption or covalent linking of the mesoporous silica particles to the substrate. Microscopy glass slides were initially functionalized by an aminosilane to which a thin layer of hyaluronic acid was covalently attached through an amide bond. A sub-monolayer of amino-functionalized mesoporous silica nanoparticles with a diameter of 400 nm were then adsorbed to the hyaluronic acid-functionalized surface by adsorption under high-shear conditions. Subsequently, the adsorbed particles were covalently linked to the hyaluronic acid through an amide bond. Corresponding films without the covalent coupling step were used as the control. Muscle stem cells attach and proliferate nicely on all films, and do internalize particles in all cases. However, the kinetics of particle internalization was clearly delayed for the covalently attached particles in comparison to physisorbed particles. Thus, the results imply that tuning of the particle-substrate interactions through linking chemistries is a promising means of achieving sequential particle-mediated drug release from films and that corresponding chemistries should also be applicable for 3D scaffold systems.

Selective elimination of the free fatty acid fraction from esterified fatty acids in rat plasma through chemical derivatization and immobilization on amino functionalized silica nano-particles

A high throughput and low cost approach to separate free fatty acids (FFAs) from phospholipid and acylglycerols (esterified fatty acids, EFAs) has been demonstrated, which may be widely used as a sample preparation method in the metabolomics and lipid research. The optimal conditions for FFAs reacting with N-hydroxysuccinimide (NHS) only need 10min at room temperature to obtain a 93.5% yield of FFAs-NHS ester. The rest 6% FFA transformed into N-cyclohexyl-fatty acid-amide which is stable to methyl esterification adopted for fatty acids analysis. 10min are taken for FFAs-NHS ester to react with amino functionalized silica nanoparticles to immobilize the FFAs. The separation of FFAs from EFAs could be carried out readily by centrifugation. The whole process including derivatization, immobilization, and centrifugation takes less than 40min. Much more accurate fatty acids composition of rat plasma EFAs could be obtained by this approach than the previous reported methods.

Amino-functionalized silica nanoparticles for improved enantiomeric separation in capillary electrophoresis using carboxymethyl-β-cyclodextrin (CM-β-CD) as a chiral selector

We report on the use of amino-modified silica nanoparticles (SiNPs) as an additive to the background electrolyte solution to enhance the chiral selectivity of in capillary electrophoresis that is induced by the presence of a small quantity of carboxymethyl-β-cyclodextrin (CM-β-CD). The modified SiNPs were characterized by transmission electron microscopy, elemental analysis and their zeta potential. The method was applied to the separation of four alkaline drugs (ephedrine, chlorpheniramine, propranolol and amlodipine). The addition of the modified SiNPs to the background electrolyte results in a distinct improvement in the separation power, especially when the capillary was pretreated with high concentration of particle suspensions prior to separation. The effects of fractions of modified SiNPs and organic modifier, of the thickness of the SiNP coating layer on the capillary wall were investigated. Under optimum experimental conditions, all the racemates investigated were separated with improved resolution, thus indicating the potential of the method in the field of enantiomeric separation.

An electrochemical immunosensing method for detecting melanoma cells

An electrochemical immunosensing method was developed to detect melanoma cells based on the affinity between cell surface melanocortin 1 receptor (MC1R) antigen and anti-MC1R antibody (MC1R-Ab). The MC1R-Abs were immobilized in amino-functionalized silica nanoparticles (n-SiNPs)-polypyrrole (PPy) nanocomposite modified on working electrode surface of screen-printed electrode (SPE). Cyclic voltammetry was employed, with the help of redox mediator ([Fe(CN)6]3−), to measure the change in anodic oxidation peak current arising due to the specific interaction between MC1R antigens and MC1R-Abs when the target melanoma cells are present in the sample. Various factors affecting the sensor performance, such as the amount of MC1R-Abs loaded, incubation time with the target melanoma cells, the presence of interfering non-melanoma cells, were tested and optimized over different expected melanoma cell loads in the range of 50–7500 cells/2.5mL. The immunosensor is highly sensitive (20cells/mL), specific, and reproducible, and the antibody-loaded electrode in ready-to-use stage is stable over two weeks. Thus, in conjunction with a microfluidic lab-on-a-chip device our electrochemical immunosensing approach may be suitable for highly sensitive, selective, and rapid detection of circulating tumor cells (CTCs) in blood samples.