A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au–thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer–AuNPs–HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1 × 10² to 1 × 10⁷ cells mL⁻¹ and high sensitivity with a low detection limit of 30 cells mL⁻¹. Furthermore, after the electrochemical detection, the activation potential of −0.9 to −1.7 V was performed to break Au–thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing.     

Selective collection and detection of MCF-7 breast cancer cells using aptamer-functionalized magnetic beads and quantum dots based nano-bio-probes

A novel strategy for selective collection and detection of breast cancer cells (MCF-7) based on aptamer–cell interaction was developed. Mucin 1 protein (MUC1) aptamer (Apt1) was covalently conjugated to magnetic beads to capture MCF-7 cell through affinity interaction between Apt1 and MUC1 protein that overexpressed on the surface of MCF-7 cells. Meanwhile, a nano-bio-probe was constructed by coupling of nucleolin aptamer AS1411 (Apt2) to CdTe quantum dots (QDs) which were homogeneously coated on the surfaces of monodispersed silica nanoparticles (SiO₂ NPs). The nano-bio-probe displayed similar optical and electrochemical performances to free CdTe QDs, and remained high affinity to nucleolin overexpressed cells through the interaction between AS1411 and nucleolin protein. Photoluminescence (PL) and square-wave voltammetric (SWV) assays were used to quantitatively detect MCF-7 cells. Improved selectivity was obtained by using these two aptamers together as recognition elements simultaneously, compared to using any single aptamer. Based on the signal amplification of QDs coated silica nanoparticles (QDs/SiO₂), the detection sensitivity was enhanced and a detection limit of 201 and 85 cells mL⁻¹ by PL and SWV method were achieved, respectively. The proposed strategy could be extended to detect other cells, and showed potential applications in cell imaging and drug delivery.     

A novel aptamer functionalized CuInS2 quantum dots probe for daunorubicin sensing and near infrared imaging of prostate cancer cells

In this paper, a novel daunorubicin (DNR)-loaded MUC1 aptamer-near infrared (NIR) CuInS₂ quantum dot (DNR–MUC1–QDs) conjugates were developed, which can be used as a targeted cancer imaging and sensing system. After the NIR CuInS₂ QDs conjugated with the MUC1 aptamer–(CGA)₇, DNR can intercalate into the double-stranded CG sequence of the MUC1–QDs. The incorporation of multiple CG sequences within the stem of the aptamers may further increase the loading efficiency of DNR on these conjugates. DNR–MUC1–QDs can be used to target prostate cancer cells. We evaluated the capacity of MUC1–CuInS₂ QDs for delivering DNR to cancer cells in vitro, and its binding affinity to MUC1-positive and MUC1-negative cells. This novel aptamer functionalized QDs bio-nano-system can not only deliver DNR to the targeted prostate cancer cells, but also can sense DNR by the change of photoluminescence intensity of CuInS₂ QDs, which concurrently images the cancer cells. The quenched fluorescence intensity of MUC1–QDs was proportional to the concentration of DNR in the concentration ranges of 33–88 nmol L⁻¹. The detection limit (LOD) for DNR was 19 nmol L⁻¹. We demonstrate the specificity and sensitivity of this DNR–MUC1–QDs probe as a cancer cell imaging, therapy and sensing system in vitro.     

Aptamer-quantum dots conjugates-based ultrasensitive competitive electrochemical cytosensor for the detection of tumor cell

A novel competitive electrochemical cytosensor was reported by using aptamer (Apt)-quantum dots (Qdots) conjugates as a platform for tumor cell recognition and detection. The complementary DNA (cDNA), aptamer and Qdots could be assembled to the gold electrode surface. When the target cells existed, they could compete with cDNA to bind with Apt-Qdots conjugates based on the specific recognition of aptamer to MUC1 protein overexpressed on the cell surface, which resulted in the denaturation of double-stranded DNA structure and the release of the Apt-Qdots conjugates from the electrode. Electrochemical stripping measurement was then employed to determine the Cd²⁺ concentration in Qdots left at the electrode. The peak current was inversely proportional to the logarithmic value of cell concentration ranging from 1.0 × 10² to 1.0 × 10⁶ cells mL⁻¹ with a detection limit of 100 cells mL⁻¹. Meanwhile, the recognition of aptamer to the target cells could be clearly observed through the strong fluorescence from Qdots. This is an example of the combination of aptamer and nanoparticles for the application of cell analysis, which is essential to cancer diagnosis and therapy.     

Horseradish peroxidase and aptamer dual-functionalized nanoprobe for the amplification detection of alpha-methylacyl-CoA racemase

Alpha-methylacyl-CoA racemase (AMACR) is over-expressed in many cancer types and can serve as a novel diagnostic biomarker. Development of convenient and sensitive detection methods of AMACR is of particular importance for cancer diagnosis. Aptamers are a type of recognition elements, which possess many advantages over antibody, making them suitable for applications in biosensing and biotechnology. In this work, we use the efficient surface modification of gold nanoparticles (AuNPs) to prepare the horseradish peroxidase (HRP) and aptamer dual-functionalized nanoprobe. The immobilization of HRP and thiol-terminated aptamer on the surface of AuNPs can be achieved through electrostatic interaction and the formation of Au–S bond, respectively. This nanoprobe, which is used as discriminating and catalytic probe, can be combined with enzyme immunoassay method to increase the detection sensitivity of AMACR. The detection limit can reach as low as 4.6 pg mL⁻¹ due to the dual signal amplification from enzymatic cycling and the high loading of enzymes on AuNPs. This sensitivity is about three orders of magnitude higher than that of AMACR aptamer based fluorescence method, which is also comparable to or one order of magnitude higher than that of ELISA. Furthermore, this method is more simple and effective, which not only avoids the conjugation between recognition element and the catalytic enzyme, but also achieves greater signal amplification. This assay could be used as a sensitive and selective platform for the detection of target protein.     

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY_0.78F₄:Yb_0.2,Tm_0.02 UCNPs modified aptamer 1 and NaY_0.28F₄:Yb_0.70,Er_0.02 UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9964), and the signal was in the range of 10¹–10⁵ cfu mL⁻¹ (R² = 0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL⁻¹ for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF₄ UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria.     

The further study on radioiodinated peptide Arg-Arg-Leu targeted to neovascularization as well as tumor cells in molecular tumor imaging

The importance of angiogenesis in tumor growth and metastasis has led to develop new imaging tracers to understand angiogenic vasculature. Based on the previous study, we further focused on the tumor molecular imaging application of the novel peptide Arginine-Arginine-Leucine (Tyr-Cys-Gly-Gly-Arg-Arg-Leu-Gly-Gly-Cys, tRRL) in this study. The cytotoxicity of raioiodinated tRRL (¹³¹I-tRRL) in HepG2 cells was assessed by tested cell viability using kit. tRRL was conjugated with fluorescein FITC to observe its binding with tumor cells and human aortic endothelial cells (HAEC) in vitro. Whole body SPECT imaging of varied tumors xenograftes was performed after intravenous injection of ¹³¹I-tRRL for 24 h in BALB/c nude mice. Compared with negative control PBS, small peptide tRRL was of non-cytotoxicity. ¹³¹I-tRRL could lead to significant cytotoxicity on HepG2 cells when the radioactivity was greater than 370 kBq. In vitro binding experiment and cellular uptake results revealed that tRRL could adhere to tumor cells besides tumor derived endothelial cells. In vivo SPECT imaging, ¹³¹I-tRRL mainly accumulated in various tumor tissues, including melanoma, liver cancer and lung cancer bearing mice. In breast cancer xenografte imaging, the tumor has no significant radionuclide accumulation at 24 h after injected of ¹³¹I-tRRL. Radioiodinated tRRL offers a noninvasive nuclear imaging method for functional molecular imaging of tumors, and may be a promising candidate carrier for tumor targeted therapy.     

Multifunctional Uniform Core–Shell Fe3O4@mSiO2 Mesoporous Nanoparticles for Bimodal Imaging and Photothermal Therapy

Multimodal imaging and simultaneous therapy is highly desirable because it can provide complementary information from each imaging modality for accurate diagnosis and, at the same time, afford an imaging‐guided focused tumor therapy. In this study, indocyanine green (ICG), a near‐infrared (NIR) imaging agent and perfect NIR light absorber for laser‐mediated photothermal therapy, was successfully incorporated into superparamagnetic Fe₃O₄@mSiO₂ core–shell nanoparticles to combine the merit of NIR/magnetic resonance (MR) bimodal imaging properties with NIR photothermal therapy. The resultant nanoparticles were homogenously coated with poly(allylamine hydrochloride) (PAH) to make the surface of the composite nanoparticles positively charged, which would enhance cellular uptake driven by electrostatic interactions between the positive surface of the nanoparticles and the negative surface of the cancer cell. A high biocompatibility of the achieved nanoparticles was demonstrated by using a cell cytotoxicity assay. Moreover, confocal laser scanning microscopy (CLSM) observations indicated excellent NIR fluorescent imaging properties of the ICG‐loaded nanoparticles. The relatively high r₂ value (171.6 mM ^?1 s^?1) of the nanoparticles implies its excellent capability as a contrast agent for MRI. More importantly, the ICG‐loaded nanoparticles showed perfect NIR photothermal therapy properties, thus indicating their potential for simultaneous cancer diagnosis as highly effective NIR/MR bimodal imaging probes and for NIR photothermal therapy of cancerous cells.     

MnO2‐Modified Persistent Luminescence Nanoparticles for Detection and Imaging of Glutathione in Living Cells and In Vivo

Persistent luminescence nanoparticles (PLNPs) hold great promise for the detection and imaging of biomolecules. Herein, we have demonstrated a novel nanoprobe, based on the manganese dioxide (MnO₂)‐modified PLNPs, that can detect and image glutathione in living cells and in vivo. The persistent luminescence of the PLNPs can be efficiently quenched by the MnO₂ nanosheets. In the presence of glutathione (GSH), MnO₂ was reduced to Mn²⁺ and the luminescence of PLNPs can be restored. The persistent luminescence property can allow detection and imaging without external excitation and avoid the background noise originating from the in situ excitation. This strategy can offer a promising platform for detection and imaging of reactive species in living cells or in vivo.     

Rapid Legionella pneumophila determination based on a disposable core–shell Fe3O4@poly(dopamine) magnetic nanoparticles immunoplatform

A novel amperometric magnetoimmunoassay, based on the use of core–shell magnetic nanoparticles and screen-printed carbon electrodes, was developed for the selective determination of Legionella pneumophila SG1. A specific capture antibody (Ab) was linked to the poly(dopamine)–modified magnetic nanoparticles (MNPs@pDA-Ab) and incubated with bacteria. The captured bacteria were sandwiched using the antibody labeled with horseradish peroxidase (Ab-HRP), and the resulting MNPs@pDA-Ab-Legionella neumophila-Ab-HRP were captured by a magnetic field on the electrode surface. The amperometric response measured at −0.15 V vs. Ag pseudo-reference electrode of the SPCE after the addition of H₂O₂ in the presence of hydroquinone (HQ) was used as transduction signal. The achieved limit of detection, without pre-concentration or pre-enrichment steps, was 10⁴ Colony Forming Units (CFUs) mL⁻¹. The method showed a good selectivity and the MNPs@pDA-Ab exhibited a good stability during 30 days. The possibility of detecting L. pneumophila at 10 CFU mL⁻¹ level in less than 3 h, after performing a membrane-based preconcentration step, was also demonstrated.     

A pH-activatable and aniline-substituted photosensitizer for near-infrared cancer theranostics

This work reports a newly designed pH-activatable and aniline-substituted aza-boron-dipyrromethene as a trifunctional photosensitizer to achieve highly selective tumor imaging, efficient photodynamic therapy (PDT) and therapeutic self-monitoring through encapsulation in a cRGD-functionalized nanomicelle. The diethylaminophenyl is introduced in to the structure for pH-activatable near-infrared fluorescence and singlet oxygen (¹O₂) generation, and bromophenyl is imported to increase the ¹O₂ generation efficiency upon pH activation by virtue of its heavy atom effect. After encapsulation, the nanoprobe can target α_vβ₃ integrin-rich tumor cells via cRGD and is activated by physiologically acidic pH for cancer discrimination and PDT. The fascinating advantage of the nanoprobe is near-infrared implementation beyond 800 nm, which significantly improves the imaging sensitivity and increases the penetration depth of the PDT. By monitoring the fluorescence decrease in the tumor region after PDT, the therapeutic efficacy is demonstrated in situ and in real time, which provides a valuable and convenient self-feedback function for PDT efficacy tracking. Therefore, this rationally designed and carefully engineered nanoprobe offers a new paradigm for precise tumor theranostics and may provide novel opportunities for future clinical cancer treatment.     

Highly Water‐Dispersible Surface‐Modified Gd2O3 Nanoparticles for Potential Dual‐Modal Bioimaging

Water‐dispersible and luminescent gadolinium oxide (GO) nanoparticles (NPs) were designed and synthesized for potential dual‐modal biological imaging. They were obtained by capping gadolinium oxide nanoparticles with a fluorescent glycol‐based conjugated carboxylate (H L ). The obtained nanoparticles (GO‐ L ) show long‐term colloidal stability and intense blue fluorescence. In addition, L can sensitize the luminescence of europium(III) through the so‐called antenna effect. Thus, to extend the spectral ranges of emission, europium was introduced into L‐ modified gadolinium oxide nanoparticles. The obtained Eu^III‐doped particles (Eu:GO‐ L ) can provide visible red emission, which is more intensive than that without L capping. The average diameter of the monodisperse modified oxide cores is about 4 nm. The average hydrodynamic diameter of the L ‐modified nanoparticles was estimated to be about 13 nm. The nanoparticles show effective longitudinal water proton relaxivity. The relaxivity values obtained for GO‐ L and Eu:GO‐ L were r₁=6.4 and 6.3 s^?1 mM ^?1 with r₂/r₁ ratios close to unity at 1.4 T. Longitudinal proton relaxivities of these nanoparticles are higher than those of positive contrast agents based on gadolinium complexes such as Gd‐DOTA, which are commonly used for clinical magnetic resonance imaging. Moreover, these particles are suitable for cellular imaging and show good biocompatibility.     

Equipping Natural Killer Cells with Specific Targeting and Checkpoint Blocking Aptamers for Enhanced Adoptive Immunotherapy in Solid Tumors

Herein, we propose an aptamer‐equipping strategy to generate specific, universal and permeable (SUPER) NK cells for enhanced immunotherapy in solid tumors. NK cells were chemically equipped with TLS11a aptamer targeting HepG2 cells and PDL1‐specific aptamer without genetic alteration. The dual aptamer‐equipped NK cells exhibited high specificity to tumor cells, resulting in higher cytokine secretion and apoptosis/necrosis compared to parental or single aptamer‐equipped NK cells. Interestingly, dual aptamer‐equipped NK cells induced remarkable upregulation of PDL1 expression in HepG2 cells, enhancing checkpoint blockade. Furthermore, in vivo intravital imaging demonstrated high infiltration of aptamer‐equipped NK cells into deep tumor region, leading to enhanced therapeutic efficacy in solid tumors. This work offers a straightforward chemical strategy to equip NK cells with aptamers, holding considerable potential for enhanced adoptive immunotherapy in solid tumors.     

18FDG conjugated magnetic nanoparticle probes: synthesis and in vitro investigations on MCF-7 breast cancer cells

¹⁸FDG conjugated magnetic iron oxide nanoparticles (MNPs) were synthesized as PET-MR hybrid imaging agent. Synthesized and characterized NPs were then applied to MCF-7 human breast cancer cells. ¹⁸FDG conjugated MNPs exhibited the cell incorporation ratio up to 30 %. As well as the characterization studies, apoptotic effects were observed depending on the cellular incorporations by the time. In conclusion, synthesized structures could have a potential as hybrid imaging agent in PET-MR imaging systems besides apoptotic effect on cancer cells.     

In situ emulsification microextraction using a dicationic ionic liquid followed by magnetic assisted physisorption for determination of lead prior to micro-sampling flame atomic absorption spectrometry

For the first time, a simple and efficient in situ emulsification microextraction method using a dicationic ionic liquid followed by magnetic assisted physisorption was presented to determine trace amounts of lead. In this method, 400 μL of 1.0 mol L⁻¹ lithium bis (trifluoromethylsulfonyl) imide aqueous solution, Li[NTf₂], was added into the sample solution containing 100 μL of 1.0 mol L⁻¹ 1,3-(propyl-1,3-diyl) bis (3-methylimidazolium) chloride, [pbmim]Cl₂, to form a water immiscible ionic liquid, [pbmim][NTf₂]₂. This new in situ formed dicationic ionic liquid was applied as the acceptor phase to extract the lead-ammonium pyrrolidinedithiocarbamate (Pb-APDC) complexes from the sample solution. Subsequently, 30 mg of Fe₃O₄ magnetic nanoparticles (MNPs) were added into the sample solution to collect the fine droplets of [pbmim][NTf₂]₂, physisorptively. Finally, MNPs were eluted by acetonitrile, separated by an external magnetic field and the obtained eluent was subjected to micro-sampling flame atomic absorption spectrometry (FAAS) for further analysis. Comparing with other microextraction methods, no special devices and centrifugation step are required. Parameters influencing the extraction efficiency such as extraction time, pH, concentration of chelating agent, amount of MNPs and coexisting interferences were studied. Under the optimized conditions, this method showed high extraction recovery of 93% with low LOD of 0.7 μg L⁻¹. Good linearity was obtained in the range of 2.5–150 μg L⁻¹ with determination coefficient (r²) of 0.9921. Relative standard deviation (RSD%) for seven repeated measurements at the concentration of 10 μg L⁻¹ was 4.1%. Finally, this method was successfully applied for determination of lead in some water and plant samples.     

Nitrogenous sesquiterpenes from the Thai marine sponge Halichondria sp.

Five nitrogenous sesquiterpenes having an isonitrile [(−)-axisonitrile-3], a formamide [(+)-axamide-3, axamide-2 and (3S^*,5R^*,6R^*,9R^*)-3-formamido-1(10)-cadinene], and an amine [(−)-halichamine] functionality were isolated from the Thai marine sponge Halichondria sp., together with two steroids, ergosterol and ergosterol peroxide. (−)-Axisonitrile-3 was isolated from the natural source for the first time, while (+)-axamide-3 and (−)-halichamine were new metabolites. The structures of these compounds were elucidated on the basis of their spectroscopic data and by chemical transformations. All sesquiterpenes were tested for their cytotoxic activity against six cancer cell lines (HeLa, HuCCA-1, A549, MOLT-3, HepG2, MDA-MB231). Only (−)-axisonitrile-3 showed strong activity to the HepG2 cell line with an IC₅₀ value of 1.3 μM.     

基于一维DNA组装磁性纳米探针的夹心型安培免疫传感器研究

采用Fe3O4(核)/ZrO2(壳)纳米磁珠(ZMPs)标记待测物识别抗体,并用HRP酶封闭和DNA链接,建立了一类新型的"珠链状"一维磁性纳米探针制备方法。将甲胎蛋白(AFP)一抗固定于纳米金修饰的玻碳电极表面,构建了免疫电极(GCE?AFP Ab1)。基于该电极和上述合成探针,通过双抗体夹心法测定免疫产物上HRP酶对过氧化脲(CP)氧化对苯二酚反应的催化电流,研制了一类基于一维纳米结构组装的夹心型安培免疫传感器。研究表明:此一维纳米结构探针不仅大大增加了酶在电极表面的富集量,成倍扩增了催化电流,显著提高了传感器的灵敏度,而且易于通过外磁场与背景液可控分离,简化了分析步骤,并提高了结果的重复性。此传感器对AFP检测的线性范围为0.01~25 mg/L;检出限达4 ng/L(3σ),并被用于人血清中痕量AFP的测定,结果满意。     

General Protocol for the Synthesis of Functionalized Magnetic Nanoparticles for Magnetic Resonance Imaging from Protected Metal–Organic Precursors

The development of magnetic nanoparticles (MNPs) with functional groups has been intensively pursued in recent years. Herein, a simple, versatile, and cost‐effective strategy to synthesize water‐soluble and amino‐functionalized MNPs, based on the thermal decomposition of phthalimide‐protected metal–organic precursors followed by deprotection, was developed. The resulting amino‐functionalized Fe₃O₄, MnFe₂O₄, and Mn₃O₄ MNPs with particle sizes of about 14.3, 7.5, and 6.6 nm, respectively, had narrow size distributions and good dispersibility in water. These MNPs also exhibited high magnetism and relaxivities of r₂=107.25 mM^?1 s^?1 for Fe₃O₄, r₂=245.75 mM^?1 s^?1 for MnFe₂O₄, and r₁=2.74 mM^?1 s^?1 for Mn₃O₄. The amino‐functionalized MNPs were further conjugated with a fluorescent dye (rhodamine B) and a targeting ligand (folic acid: FA) and used as multifunctional probes. Magnetic resonance imaging and flow‐cytometric studies showed that these probes could specifically target cancer cells overexpressing FA receptors. This new protocol opens a new way for the synthesis and design of water‐soluble and amino‐functionalized MNPs by an easy and versatile route.     

A Highly Sensitive Resonance Scattering Spectral Assay for Hg2+ Based on the Aptamer-Modified AuRu Nanoparticle-NaClO3-NaI-Cationic Surfactant Catalytic Reaction

Gold ruthenium (AuRu) nanoparticles were modified by single strand DNA (ssDNA) to prepare an aptamer AuRu nanoprobe (AuRussDNA) for Hg²⁺. The nanoprobe reacted with Hg²⁺ to form double-stranded T-Hg²⁺-T mismatches, and the released AuRu nanoparticles aggregated to big particles, which induced an increase in the resonance scattering (RS) signal at 592 nm. The RS signal was linear to the concentration of Hg²⁺ in the range of 0.0067–3.3 nmol L^?1. Using the AuRussDNA in filtration solution as a catalyst, a new catalytic RS assay was proposed for detection of trace Hg²⁺. This method was applied for the determination of Hg²⁺ in real samples.     

Synthesis of Cholesterol-Conjugated Magnetic Nanoparticles for Purification of Human Paraoxonase 1

Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe₃O₄ nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm⁻¹, and at 2,334.9 cm⁻¹ confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg⁻¹ protein, and the purification fold was 515 with 73% yield. The K _m values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg⁺², Mn⁺², glycerol, (NH₄)₂SO₄, PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb⁺², Co⁺², Zn²⁺, ethanol, β-mercaptoethanol, and acetone reduced the activity while Ni²⁺, Cd²⁺, Cu²⁺, iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml⁻¹, respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.