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1.
Food Chem ; 366: 130560, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34284183

RESUMO

The colorimetric method can determine the initial results even by the naked eyes, but its main challenge for antibiotics detection in food at present is the relatively low sensitivity. Herein, an ultrasensitive colorimetric biosensor based on G-quadruplex DNAzyme was firstly proposed for the rapid detection of trace tetracycline antibiotics like tetracycline, oxytetracycline, chlortetracycline and doxycycline. DNAzyme composed of hemin and G-quadruplex has peroxidase-like activity, and tetracyclines can combine with hemin to form a stable complex and reduce catalytic activity, making the color of solution changes from yellow to green. The limits of detection (LOD) of the proposed colorimetric biosensor for tetracyclines is determined as low as 3.1 nM, which is lower than most of the other colorimetric methods for antibiotics detection. Moreover, the average recovery range of tetracyclines in actual samples is from 89% to 99%, indicating that such strategy may has bright application prospects for tetracyclines detection in foods.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , Tetraciclinas/análise , Antibacterianos , Colorimetria , Quadruplex G , Hemina , Peroxidase , Peroxidases
2.
Biosens Bioelectron ; 197: 113806, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34808591

RESUMO

Photocathodic biosensor has great capability in anti-interference from reductive substances, however, the low signal intensity of photoactive species with inferior detection sensitivity restricts its wide application. In this work, the P3HT-PbS nanocomposites were synthesized as signal tags, by integrating with target-trigger generated hemin/G-quadruplex nanotail as bi-enhancer to significantly apmplify the photocurrent, an ultrasensitive photocathodic biosensor was proposed for detection of ß2-microglobulin (ß2-MG). Impressively, P3HT with cathode signal is an attractive polymer consisted of substantial thiophene groups with high absorption coefficient and mobility of photo-generated holes, which could anchor with the PbS dots as sensitizer, providing a high charge mobility and strong photosensitivity. More importantly, target-trigger generated hemin/G-quadruplexes could accept the electron from illuminated photoactive species through the conversion of Fe(III)/Fe(II) in hemin, effectively reducing charge recombination rate as well as accelerating the generation of electron acceptor O2 in the assistant of H2O2. Moreover, hemin/G-quadruplexes inherited the HRP mimicking catalytic capability that further improved the produce of plentiful O2. As a result, PEC cathode signal was significantly enhanced for sensitive analysis of ß2-MG protein with a good detection range of 0.1 pg/mL to 100 ng/mL. It would provide a path for establishing PEC platform with excellent anti-interference ability and extend the application of photoelectrochemical (PEC) biosensor in bioanalysis and early disease diagnosis.


Assuntos
Técnicas Biossensoriais , Quadruplex G , Nanocompostos , Técnicas Eletroquímicas , Compostos Férricos , Hemina , Peróxido de Hidrogênio , Limite de Detecção
3.
Talanta ; 238(Pt 1): 122995, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34857328

RESUMO

A highly sensitive and specific ELISA-like chemiluminescence method for detection of fibrin has been developed. In the sensing platform, the homing peptide (CREKA), as recognition molecule, which can specially recognize the fibrin on microtiter plate, combined with G-quadruplex-based DNAzyme to form the probe of G-quadruplex-hemin DNAzyme-CREKA. After the sample solution was coated on the plates, the probe was crosslinked with fibrin through the interaction of CREKA and fibrin. Finally, luminol-H2O2 chemiluminesecence (CL) reaction was exploited for quantitative analysis of fibrin. The liner range for fibrin detection was from 0.112 pmol L-1 to 5.6 pmol L-1 with the detection limit of fibrin as low as 0.04 pmol L-1, based on a signal-to-noise ratio (S/N) of 3. Furthermore, on the basis of the high amplification efficiency of the rolling circle amplification (RCA) reaction, the method enabled to analyze fibrin with a detection limit corresponding to 0.06 fmol L-1, whose sensitivity increased 3 orders of magnitude than that of above method in the absence of RCA reaction. In particular, combined with the separation and washing steps of ELISA, the proposed method possessed higher selectivity, high-throughput and low cost, which shows promise for applications in clinical diagnosis.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , DNA Catalítico/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibrina , Hemina , Peróxido de Hidrogênio , Limite de Detecção , Peptídeos
4.
Food Chem ; 372: 131253, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34818728

RESUMO

To investigate the effect of pH on lipid oxidation of chicken muscle, chicken hemolysates were added to washed chicken muscles to analyze lipid oxidation at pH 5.7, 6.3, and 7.2. The results showed that with a blue shift of the Soret peak, oxyhemoglobin gradually transformed to methemoglobin during storage, the shape of porphyrin rings of heme in fluorescence electron microscopy changed from round to trail-like structure. These changes were more significant at low pH. Comparing hemoglobin (Hb) structure, the distance ofamino acids between the E10 of lysine and metHb-7-propionate groups is longer at pH 5.7 than other pHs, which makes solvent easily enter the heme cavity, leading tothe severe destruction of Hb. The linear correlation between color and lipid oxidation also further confirmed that the increased oxidation of chicken Hb causes more rapid lipid oxidation in pH 5.7 than the other 2 pHs (p < 0.05).


Assuntos
Galinhas , Hemina , Animais , Galinhas/metabolismo , Hemina/metabolismo , Hemoglobinas/metabolismo , Lipídeos , Músculos/metabolismo , Oxirredução
5.
J Colloid Interface Sci ; 607(Pt 1): 470-478, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34509729

RESUMO

Effective electronic interactions between molecular catalysts and supports are critical for heterogeneous enzyme mimics, yet they are frequently neglected in most catalyst designs. Taking the enzyme mimics of hemin immobilized on graphdiyne (Hemin-GDY) as an example, we explicate for the first time the underlying role of GDY as a co-catalyst. Based on the robust conjugation between GDY and hemin, the delocalized π-electrons in GDY act as a ligand for Fe ions so that the orbital interactions including electron transport from GDY â†’ Fe can induce the formation of an electron-rich Fe center and an electron-deficient π-electron conjugated system. This mechanism was validated by electron paramagnetic resonance (EPR), Raman spectroscopy, and DFT calculations. Moreover, both EPR spetra and Lineweaver-Burk plots revealed that Hemin-GDY could efficiently catalyze the decomposition of hydrogen peroxide (H2O2) to produce hydroxyl radical (•OH) and superoxide anion (O2•-) by a ping-pong type catalytic mechanism, and particularly, the catalytic activity was increased by 2.3-fold comparing to that of hemin immobilized on graphene (Hemin-GR). In addition, Hemin-GDY with the exceptional activity and stability was demonstrated for efficient catalytic degradation of organic pollutants under acidic conditions. Collectively, this work provides a theoretical basis for the design of GDY supported catalysts and renders great promises of the GDY based enzyme mimics.


Assuntos
Grafite , Biomimética , Hemina , Peróxido de Hidrogênio , Peroxidase , Peroxidases
6.
Biosens Bioelectron ; 195: 113630, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34536724

RESUMO

Nitric oxide as a signal molecule participates in a variety of physiological and pathological processes but its real-time detection in cell assays still faces challenging because of the trace amount, short half-life and easy conversion to other substances. We report here a rational design by assembling highly π-conjugated and small capacitive gaphdiyne (GDY) with a coordination complex of hemin (HEM) into a molecularly assembled material of GDY/HEM to achieve ultrafast and real-time monitoring of nitric oxide in cell assays. GDY comprising alkynyl C atoms can hybridize with the HEM to enable strong π-π interaction and atomic dispersion of iron sites while avoiding the formation of catalytically inactive dimer for the HEM. These characteristics make the GDY/HEM an excellent sensing material towards nitric oxide, which has an ultrafast response time of 0.95 s, a low detection limit of 7 nM and long linear range up to 151.38 µΜ. The GDY/HEM realizes real-time monitoring nitric oxide released from cancer and normal cells, demonstrating its capability for cell analysis.


Assuntos
Técnicas Biossensoriais , Grafite , Hemina , Óxido Nítrico
7.
Analyst ; 146(23): 7284-7293, 2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34749389

RESUMO

Graphdiyne oxide (GDYO) is a novel type of two-dimensional carbon allotrope nanomaterial consisting of a large conjugated system and excellent chemical stability. To date, application of GDYO as a nanozyme in biosensing has been rarely reported. In this study, a novel ultrasensitive colorimetric bioassay was constructed using a hemin/GDYO nanocomposite (H/GDYO) as a new nanozyme with superior peroxidase-like activity for the detection of H2O2 and glucose. It was discovered that H/GDYO exhibited 6-fold higher peroxidase-like activity than pure hemin. Catalytic kinetic analysis showed that H/GDYO had a much higher affinity for H2O2 and glucose than that of hemin. The designed colorimetric bioassay displayed excellent sensitivity for H2O2 and glucose detection with a wide linear range of 0.015-0.5 mM and 0.1-10 mM, respectively, while the limit of detection (LOD) was as low as 4.39 µM and 38 µM, respectively. Moreover, it was successfully applied for the analysis of H2O2 in milk and glucose in real human serum samples with acceptable recoveries. Importantly, the developed colorimetric bioassay shows good agreement with the results obtained from a commercial blood glucose meter. We believe that the proposed method could provide a promising prospect for medical diagnosis and biotechnology.


Assuntos
Técnicas Biossensoriais , Nanocompostos , Bioensaio , Biomimética , Colorimetria , Grafite , Hemina , Humanos , Peróxido de Hidrogênio , Cinética , Óxidos , Peroxidase/metabolismo
8.
Sci Rep ; 11(1): 21462, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34728736

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 (COVID-19). More than 143 million cases of COVID-19 have been reported to date, with the global death rate at 2.13%. Currently, there are no licensed therapeutics for controlling SARS-CoV-2 infection. The antiviral effects of heme oxygenase-1 (HO-1), a cytoprotective enzyme that inhibits the inflammatory response and reduces oxidative stress, have been investigated in several viral infections. To confirm whether HO-1 suppresses SARS-CoV-2 infection, we assessed the antiviral activity of hemin, an effective and safe HO-1 inducer, in SARS-CoV-2 infection. We found that treatment with hemin efficiently suppressed SARS-CoV-2 replication (selectivity index: 249.7012). Besides, the transient expression of HO-1 using an expression vector also suppressed the growth of the virus in cells. Free iron and biliverdin, which are metabolic byproducts of heme catalysis by HO-1, also suppressed the viral infection. Additionally, hemin indirectly increased the expression of interferon-stimulated proteins known to restrict SARS-CoV-2 replication. Overall, the findings suggested that HO-1, induced by hemin, effectively suppressed SARS-CoV-2 in vitro. Therefore, HO-1 could be potential therapeutic candidate for COVID-19.


Assuntos
Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Heme Oxigenase-1/metabolismo , Hemina/uso terapêutico , Animais , Antivirais/química , Antivirais/farmacologia , COVID-19/virologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Hemina/química , Hemina/farmacologia , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Regulação para Cima/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacos
9.
Analyst ; 146(21): 6470-6473, 2021 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-34609387

RESUMO

A novel and low-cost DNAzyme, Ni/Fe layered double hydroxide (LDH) nanosheet/G-quadruplex (without hemin) with enhanced peroxidase-mimic activity was designed. The catalytic mechanism was investigated. The detection of Cu(II) in actual serum samples could be realized sensitively via this efficient DNAzyme-based method.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , DNA Catalítico/metabolismo , Hemina , Hidróxidos , Peroxidase , Peroxidases
10.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639197

RESUMO

TRPV1 mediates pain occurring during sickling episodes in sickle cell disease (SCD). We examined if hemin, a porphyrin released during intravascular hemolysis modulates TRPV1. Calcium imaging and patch clamp were employed to examine effects of hemin on mouse dorsal root ganglion (DRG) neurons and HEK293t cells expressing TRPV1 and TRPA1. Hemin induced a concentration-dependent calcium influx in DRG neurons which was abolished by the unspecific TRP-channel inhibitor ruthenium red. The selective TRPV1-inhibitor BCTC or genetic deletion of TRPV1 only marginally impaired hemin-induced calcium influx in DRG neurons. While hTRPV1 expressed in HEK293 cells mediated a hemin-induced calcium influx which was blocked by BCTC, patch clamp recordings only showed potentiated proton- and heat-evoked currents. This effect was abolished by the PKC-inhibitor chelerythrine chloride and in protein kinase C (PKC)-insensitive TRPV1-mutants. Hemin-induced calcium influx through TRPV1 was only partly PKC-sensitive, but it was abolished by the reducing agent dithiothreitol (DTT). In contrast, hemin-induced potentiation of inward currents was not reduced by DTT. Hemin also induced a redox-dependent calcium influx, but not inward currents on hTRPA1. Our data suggest that hemin induces a PKC-mediated sensitization of TRPV1. However, it also acts as a photosensitizer when exposed to UVA-light used for calcium imaging. The resulting activation of redox-sensitive ion channels such as TRPV1 and TRPA1 may be an in vitro artifact with limited physiological relevance.


Assuntos
Gânglios Espinais/metabolismo , Hemina/farmacologia , Neurônios/metabolismo , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/fisiologia , Animais , Cálcio/metabolismo , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Canal de Cátion TRPA1/efeitos dos fármacos , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética
11.
Analyst ; 146(19): 5951-5961, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34490872

RESUMO

The stability, repeatability and sensitivity of an electrochemical biosensor material are closely connected with the dispersibility of metal organic frameworks (MOFs) in aqueous media. Herein, a nanocomposite based on Cu-MOF/hemin, which is not only highly water-soluble but also simple and efficient in synthesis, was used for the construction of a non-enzymatic sensor to detect hydrogen peroxide (H2O2). The Cu-MOF/hemin was characterized via scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS)-mapping, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS) and thermal gravimetric analysis (TGA), which indicate that hemin and the Cu-MOF were successfully combined. As a H2O2 electrochemical biomimetic enzyme, the Cu-MOF/hemin exhibited excellent electrocatalytic performance, which was confirmed by the electrochemical experiments and chromogenic reactions, and the possible mechanism of the reactions has been deduced. The electrochemical sensor based on the biomimetic enzyme exhibited an extended linear detection range from 0.01-5.0 mM (R = 0.998), low detection limit of 4.14 µM, and high selectivity and stability under the optimized conditions. More importantly, the practical application ability of the sensor was verified by the test of H2O2 in human serum samples and it could be used for the real-time detection of H2O2 released from living cells with satisfactory results. Therefore, this novel nanocomposite has certain potential in preparing electrochemical sensing platforms for nonenzymatic biosensing and provides a new method for clinical diagnosis and real-time monitoring.


Assuntos
Hemina , Estruturas Metalorgânicas , Biônica , Técnicas Eletroquímicas , Humanos , Peróxido de Hidrogênio , Espectroscopia de Infravermelho com Transformada de Fourier
12.
Inorg Chem ; 60(19): 14515-14519, 2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34505770

RESUMO

Nanozyme is a class of artificial materials that possess enzyme-like activities and can overcome limitations of natural enzymes. However, controllability of the active sites, uniformity of the particles, and dispersion in the physiological media are still challenging for nanomaterial-based nanozymes. In this work, a protein-based nanozyme has been constructed by the encapsulation of hemin into the nanocavity of a recombinant human heavy chain ferritin (Ftn), generating a monodispersed peroxidase-mimetic nanozyme (hemin@Ftn). Hemin@Ftn possesses high peroxidase catalytic activity and high tolerance to the harsh environmental conditions, such as high temperature and chemical denaturant. Remarkably, hemin@Ftn can act as a colorimetric probe for the detection of tumor cells because it can selectively catalyze reactions in tumor cells. This protein-based nanozyme bridges the gap between natural enzymes and nanomaterial-based nanozymes by the incorporation of a catalytically active prosthetic group into a highly stable Ftn.


Assuntos
Ferritinas/química , Hemina/química , Nanoestruturas/química , Animais , Linhagem Celular , Humanos , Camundongos , Espectrofotometria Ultravioleta
13.
Nanoscale ; 13(31): 13231-13240, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477731

RESUMO

Although artemisinin (ART) has shown initial promise in cancer therapy, its therapeutic efficacy is limited by its low tumor inhibitory efficacy and unfavorable distribution. Considering the important role of heme in the specific parasite-killing effect of ART, we designed a liposomal nanostructure self-assembled from hemin-lipid (Hemesome) to co-deliver ART and hemin for cancer therapy. The synergistic chemotherapeutic and immunotherapeutic effects of hemin and ART were demonstrated both in vitro and in vivo. The liposome-like structure was relatively stable in the blood circulation and gastrointestinal tract environment, but dissociated in the tumor cell environment. The folic acid (FA) modification not only increased their efficiency for transport across the epithelium, but also increased their tumor accumulation. In mouse models, following oral administration of FA-Hemesome-ART nanoparticles (5 mg kg-1 ART in total) every other day and intraperitoneal injection with a programmed death-ligand 1 antibody (aPD-L1, 70 µg per mouse in total), MC38 tumors were completely inhibited within 30 days. The cured mice remained tumor-free 30 days after rechallenging them with another inoculation of MC38 cells due to the strong immune memory effect.


Assuntos
Artemisininas , Nanopartículas , Neoplasias , Animais , Linhagem Celular Tumoral , Hemina , Imunoterapia , Lipídeos , Camundongos , Neoplasias/tratamento farmacológico
14.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34576050

RESUMO

Intracerebral hemorrhage (ICH) occurs when brain blood vessels rupture, causing inflammation and cell death. 2-Fucosyllactose (2FL), a human milk oligosaccharide, has potent antiapoptotic and anti-inflammatory effects. The purpose of this study was to examine the protective effect of 2FL in cellular and rodent models of ICH. Hemin was added to a primary rat cortical neuronal and BV2 microglia coculture to simulate ICH in vitro. IBA1 and MAP2 immunoreactivities were used to determine inflammation and neuronal survival. Hemin significantly increased IBA1, while it reduced MAP2 immunoreactivity. 2FL significantly antagonized both responses. The protective effect of 2FL was next examined in a rat ICH model. Intracerebral administration of type VII collagenase reduced open-field locomotor activity. Early post-treatment with 2FL significantly improved locomotor activity. Brain tissues were collected for immunohistochemistry and qRT-PCR analysis. 2FL reduced IBA1 and CD4 immunoreactivity in the lesioned striatum. 2FL downregulated the expression of ER stress markers (PERK and CHOP), while it upregulated M2 macrophage markers (CD206 and TGFß) in the lesioned brain. Taken together, our data support that 2FL has a neuroprotective effect against ICH through the inhibition of neuroinflammation and ER stress. 2FL may have clinical implications for the treatment of ICH.


Assuntos
Proteínas de Ligação ao Cálcio/genética , AVC Hemorrágico/tratamento farmacológico , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Trissacarídeos/farmacologia , Animais , Linhagem Celular , Técnicas de Cocultura , Colagenases/toxicidade , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hemina/toxicidade , AVC Hemorrágico/induzido quimicamente , AVC Hemorrágico/genética , AVC Hemorrágico/patologia , Humanos , Locomoção/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/patologia , Leite Humano/química , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Ratos , Trissacarídeos/química
15.
Biosens Bioelectron ; 192: 113547, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34385013

RESUMO

Herein, a photocurrent polarity switching platform for highly selective assay of mucin 1 (MUC1) was developed based on target-induced hemin transfer from ZrO2 hollow spheres (ZrO2 HSs) to G-quadruplex nanowires (G wires). In this system, SiO2 spheres were used as templates to synthesize the uniform and mesoporous ZrO2 HSs. As nanocontainers, ZrO2 HSs could load hemin in its cavity via pores. Then, the aptamers of MUC1, as bio-gates, blocked the pores of ZrO2 HSs based on the specific binding of Zr4+ and the phosphate groups of aptamer. In the presence of MUC1, the aptamer could specifically recognize and bind with MUC1, and then leave away from the surface of ZrO2 HSs, which resulted in the opening of the bio-gates and releasing of hemin. Assisted with the G wires formed on the Au NPs/In2S3/ITO, the released hemin was captured on the electrode through the formation of hemin/G-quadruplex structure, leading to the switch of the photocurrent polarity of the electrode from anodic photocurrent to cathodic photocurrent. The proposed photoelectrochemical biosensor showed outstanding performance for MUC1 assay with high selectivity, wide linear response range (1 fg mL-1 -10 ng mL-1) and lower detection limit (0.48 fg mL-1). And the strategy could be easily extended to a triple-mode detection of MUC1 because the hemin/G-quadruplex structure was widely used in electrochemical and colorimetric methods as a hydrogen peroxide mimetic enzyme, which might provide wide applications in biological or clinical studies.


Assuntos
Técnicas Biossensoriais , DNA Catalítico , Quadruplex G , Nanofios , DNA Catalítico/metabolismo , Técnicas Eletroquímicas , Hemina , Limite de Detecção , Mucina-1 , Dióxido de Silício
16.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360665

RESUMO

In this work we examined the properties of thrombin-binding aptamer (TBA) modified by the introduction of inversion of polarity sites (IPS) in order to assess the effect of modification on the activation of TBA to serve as DNAzyme with peroxidase-like activity. Two oligonucleotides were designed to possess one (IPS1) or three (IPS2) inversion sites. TBA typically forms antiparallel G-quadruplexes with two G-tetrads, which exhibits very low DNAzyme peroxidise activity. DNAzyme activity is generally attributed to parallel G-quadruplexes. Hence, inversion of polarity was introduced in the TBA molecule to force the change of G-quadruplex topology. All oligonucleotides were characterized using circular dichroism and UV-Vis melting profiles. Next, the activity of the DNAzymes formed by studied oligonucleotides and hemin was investigated. The enhancement of peroxidase activity was observed when inversion of polarity was introduced. DNAzyme based on IPS2 showed the highest peroxidase activity in the presence of K+ or NH4+ ions. This proves that inversion of polarity can be used to convert a low-activity DNAzyme into a DNAzyme with high activity. Since TBA is known for its anticoagulant properties, the relevant experiments with IPS1 and IPS2 oligonucleotides were performed. Both IPS1 and IPS2 retain some anticoagulant activity in comparison to TBA in the reaction with fibrinogen. Additionally, the introduction of inversion of polarity makes these oligonucleotides more resistant to nucleases.


Assuntos
Anticoagulantes/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , DNA Catalítico/metabolismo , Fibrinogênio/metabolismo , Quadruplex G , Hemina/metabolismo , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Humanos , Modelos Moleculares
17.
Molecules ; 26(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34443603

RESUMO

Abnormal levels of reduced glutathione (GSH) and glutathione reductase (GR) are usually related to a variety of diseases, so it is of great significance to determine the GSH concentration and GR activity. We herein develop a smartphone-assisted colorimetric biosensor for the detection of GSH and GR activity in human serum and mouse liver using hemin/G-quadruplex DNAzyme. Firstly, an obvious color change from colorless to green can be observed, owing to the high peroxidase-like activity of hemin/G-quadruplex DNAzyme toward 2,2'-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS). With the addition of GSH or GR, the H2O2-mediated oxidation of ABTS catalyzed by hemin/G-quadruplex DNAzyme is significantly inhibited, resulting in remarkable color fading. Therefore, the detection of GSH and GR activity can be achieved by observing the color transition or measuring the absorbance at 420 nm. The detection limit was estimated to be as low as 0.1 µM and 10 µU/mL for GSH and GR, respectively. More interestingly, the RGB values of the sensing system can be identified by the smartphone application (APP, color collect), which makes it an ideal format for on-site determination and point-of-care testing (POCT). In addition, the proposed method shows excellent selectivity and acceptable applicability for the determination of GSH concentration and GR activity in human serum samples and mouse liver tissues, which might hold great application potential in clinical diagnosis and drug screening.


Assuntos
Técnicas Biossensoriais/métodos , DNA Catalítico/metabolismo , Glutationa Redutase/sangue , Glutationa/sangue , Hemina/metabolismo , Fígado/metabolismo , Smartphone , Animais , Colorimetria , DNA Catalítico/química , Quadruplex G , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Humanos , Camundongos , Oxirredução
18.
Biomolecules ; 11(8)2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34439841

RESUMO

The mitochondrial 2-oxoglutarate carrier (OGC), isolated and purified from rat brain mitochondria, was reconstituted into proteoliposomes to study the interaction with hemin, a porphyrin derivative, which may result from the breakdown of heme-containing proteins and plays a key role in several metabolic pathways. By kinetic approaches, on the basis of the single binding centre gated pore mechanism, we analyzed the effect of hemin on the transport rate of OGC in uptake and efflux experiments in proteoliposomes reconstituted in the presence of the substrate 2-oxoglutarate. Overall, our experimental data fit the hypothesis that hemin operates a competitive inhibition in the 0.5-10 µM concentration range. As a consequence of the OGC inhibition, the malate/aspartate shuttle might be impaired, causing an alteration of mitochondrial function. Hence, considering that the metabolism of porphyrins implies both cytoplasmic and mitochondrial processes, OGC may participate in the regulation of porphyrin derivatives availability and the related metabolic pathways that depend on them (such as oxidative phosphorylation and apoptosis). For the sake of clarity, a simplified model based on induced-fit molecular docking supported the in vitro transport assays findings that hemin was as good as 2-oxoglutarate to bind the carrier by engaging specific ionic hydrogen bond interactions with a number of key residues known for participating in the similarly located mitochondrial carrier substrate binding site.


Assuntos
Encéfalo/metabolismo , Hemina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Ligação Proteica , Proteolipídeos/metabolismo , Ratos
19.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445381

RESUMO

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.


Assuntos
Hemina/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Lacase/metabolismo , Albumina Sérica Humana/química , Tirosina/química , Sítios de Ligação , Biocatálise , Química Click , Sistemas de Liberação de Medicamentos , Humanos , Ibuprofeno/química , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Albumina Sérica Humana/metabolismo
20.
Int J Mol Sci ; 22(16)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34445741

RESUMO

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.


Assuntos
COVID-19/etiologia , Hemoglobinas/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , COVID-19/sangue , Hemina/metabolismo , Hemoglobinas/ultraestrutura , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Proteômica , Protoporfirinas/metabolismo , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/ultraestrutura , Proteínas Estruturais Virais/ultraestrutura , Ligação Viral , Replicação Viral
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