Engulfment of Hb-activated platelets differentiates monocytes into pro-inflammatory macrophages in PNH patients.

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)-activated platelets, classical monocytes differentiated into pro-inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb-activated platelets, monocytes differentiate into M1-macrophages under M1-polarization stimulus (GM-CSF, IFN-γ + LPS). When grown under M2-polarization stimulus (M-CSF, IL-4 + IL13), the cells exhibited an M1-like phenotype, secreted elevated levels of pro-inflammatory cytokines including TNF-α and IL-1ß, and displayed loss of the secretion of cytokine such as IL-10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho-STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte-platelet aggregates in circulation and accumulation of CD80high macrophages in thioglycollate-induced inflamed peritoneum. The spleen of the mice was also populated by CD80high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb-activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.

Development of pro-inflammatory phenotype in monocytes after engulfing Hb-activated platelets in hemolytic disorders.

Monocytes and macrophage combat infections and maintain homeostatic balance by engulfing microbes and apoptotic cells, and releasing inflammatory cytokines. Studies have described that these cells develop anti-inflammatory properties upon recycling the free-hemoglobin (Hb) in hemolytic conditions. While investigating the phenotype of monocytes in two hemolytic disorders-paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD), we observed a high number of pro-inflammatory (CD14+CD16hi) monocytes in these patients. We further investigated in vitro the phenotype of these monocytes and found an estimated 55% of CD14+ cells were transformed into the CD14+CD16hi subset after engulfing Hb-activated platelets. The CD14+CD16hi monocytes, which were positive for both intracellular Hb and CD42b (platelet marker), secreted significant amounts of TNF-α and IL-1ß, unlike monocytes treated with only free Hb, which secreted more IL-10. We have shown recently the presence of a high number of Hb-bound hyperactive platelets in patients with both diseases, and further investigated if the monocytes engulfed these activated platelets in vivo. As expected, we found 95% of CD14+CD16hi monocytes with both intracellular Hb and CD42b in both diseases, and they expressed high TNF-α. Furthermore our data showed that these monocytes whether from patients or developed in vitro after treatment with Hb-activated platelets, secreted significant amounts of tissue factor. Besides, these CD14+CD16hi monocytes displayed significantly decreased phagocytosis of E. coli. Our study therefore suggests that this alteration of monocyte phenotype may play a role in the increased propensity to pro-inflammatory/coagulant complications observed in these hemolytic disorders-PNH and SCD.

An electrochemical sensor for detection of neurotransmitter-acetylcholine using metal nanoparticles, 2D material and conducting polymer modified electrode.

An essential biological sensor for acetylcholine (ACh) detection is constructed by immobilizing enzymes, acetylcholinesterase (AChE) and choline oxidase (ChO), on the surface of iron oxide nanoparticles (Fe2O3NPs), poly(3,4-ethylenedioxythiophene) (PEDOT)-reduced graphene oxide (rGO) nanocomposite modified fluorine doped tin oxide (FTO). The qualitative and quantitative measurements of nanocomposites properties were accomplished by scanning electron microscope (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). This prepared biological sensor delineated a wide linear range of 4.0nM to 800µM with a response time less than 4s and detection limit (based on S/N ratio) of 4.0nM. The sensor showed perfect sensitivity, excessive selectivity and stability for longer period of time during storage. Besides its very high-sensitivity, the biosensor has displayed a low detection limit which is reported for the first time in comparison to previously reported ACh sensors. By fabricating Fe2O3NPs/rGO/PEDOT modified FTO electrode for determining ACh level in serum samples, the applicability of biosensor has increased immensely as the detection of the level neurotransmitter is first priority for patients suffering from memory loss or Alzheimer's disease (AD).

HbS Binding to GP1bα Activates Platelets in Sickle Cell Disease.

Intravascular hemolysis increases the risk of thrombosis in hemolytic disorders. Our previous study showed that the binding of adult hemoglobin (HbA) to glycoprotein (GP) 1bα induced the activation of platelets. The elevated plasma Hb or platelet surface bound Hb positively correlated with platelet activation in patients with paroxysmal nocturnal hemoglobinuria (PNH). Furthermore, this study shows that the sickle Hb [HbS, occurs due to single nucleotide polymorphism at A>T of ß-globin gene of Hb and causes sickle cell disease (SCD)] also bound to GP1bα and activated platelets in a concentration-dependent manner. The HbS bound to glycocalicin (extramembranous part of GP1bα) with KD ~ 10.46 ± 3 µM. HbS induced phosphorylation of signaling adapter proteins, such as Lyn, PI3K, Akt and ERK in platelets, and also increased the surface expression of platelet activation markers such as P-selectin (10.7 fold) and PAC1 binding (10.4 fold) in platelet surface in a concentration-dependent manner. HbS also increased the platelet microparticle-generation (4.7 fold) and thrombus-formation (4.3 fold) in a concentration-dependent manner. An elevated level of extracellular Hb in plasma correlated directly with platelet activation markers such as P-selectin (r = 0.7947), PAC1 binding (r = 0.5914) on platelet surface and plasma levels of platelet-derived microparticles (r = 0.7834) in patients with SCD. Our study therefore suggests that the HbS-induced platelet activation may play a crucial role in intravascular clot formation observed in SCD patients characterized by high propensity to vascular occlusion and hypercoagulable states.

Hemoglobin interaction with GP1bα induces platelet activation and apoptosis: a novel mechanism associated with intravascular hemolysis.

Intravascular hemolysis increases the risk of hypercoagulation and thrombosis in hemolytic disorders. Our study shows a novel mechanism by which extracellular hemoglobin directly affects platelet activation. The binding of Hb to glycoprotein1bα activates platelets. Lower concentrations of Hb (0.37-3 µM) significantly increase the phosphorylation of signaling adapter proteins, such as Lyn, PI3K, AKT, and ERK, and promote platelet aggregation in vitro. Higher concentrations of Hb (3-6 µM) activate the pro-apoptotic proteins Bak, Bax, cytochrome c, caspase-9 and caspase-3, and increase platelet clot formation. Increased plasma Hb activates platelets and promotes their apoptosis, and plays a crucial role in the pathogenesis of aggregation and development of the procoagulant state in hemolytic disorders. Furthermore, we show that in patients with paroxysmal nocturnal hemoglobinuria, a chronic hemolytic disease characterized by recurrent events of intravascular thrombosis and thromboembolism, it is the elevated plasma Hb or platelet surface bound Hb that positively correlates with platelet activation.

Type-2 diabetes: Current understanding and future perspectives.

The rapid outbreak of type-2 diabetes is one of the largest public health problems around the globe. Particularly, the developing nations are becoming the epicenters of cardiometabolic disorders owing to the change in lifestyle and diet preference besides genetic predisposition. Diabetes has become a major independent risk factor for cardiovascular diseases in South Asian countries including India. The pathogenesis of type-2 diabetes primarily initiates with inadequacy of pancreatic islet ß-cells to respond to chronic fuel surfeit and hence causing glycemic load, insulin resistance, and obesity. Urban Indian life is threatened with unhealthy high calorie diet and sedentary habits, and thus impairing the metabolic status of "thin-fat Indians" and rendering them more vulnerable to metabolic disorders. Furthermore, the metabolic dysfunction may be triggered off quite early in life due to poor maternal health and impairment in intrauterine programming and, particularly in rural India. The impaired fetal development affects the health status in later stage of life by promoting obesity, insulin resistance, type-2 diabetes, and cardiovascular complications. Therefore, the preventive and therapeutic approaches focus on a holistic strategy to improve maternal and child health, promote balanced diet and physical exercise in combination with pharmacological intervention of reducing/checking hyperglycemia, obesity, and cardiovascular complications. This review summarizes the epidemiology, mechanisms, and risk factors for diabetes and cardiovascular disorders with a focus on the Indian subcontinent.

Determination of glycated hemoglobin with special emphasis on biosensing methods.

The glycated hemoglobin (HbA1c) level in blood is a measure of long-term glycemic status in patients with diabetes mellitus. Current clinical methods for determination of the HbA1c level include electrophoresis/electroendosmosis, ion exchange chromatography, high-performance liquid chromatography, boronate affinity chromatography, immunoassay, and liquid chromatography-tandem mass spectroscopy in addition to fluorometry and colorimetry. These methods have certain drawbacks such as being complex, time-consuming, and requiring expensive apparatus and trained persons to operate. These drawbacks were overcome by biosensing methods. We review these biosensors, which are based on (i) measurement of electrons, that is, current generated from splitting of hydrogen peroxide released during oxidation of fructosyl valine by immobilized fructosyl amino acid oxidase, which is directly proportional to HbA1c concentration, and (ii) direct measurement of HbA1c by some specific reaction. HbA1c biosensors work optimally within 4 to 1800 s, between pH 7.0 and 9.0 and between 25 and 45 °C, and in the range of 1 to 10,000 µM, with a detection limit between 20 and 500 µM and sensitivity between 4.6 nA and 21.5 µA mM⁻¹ cm⁻² and stable over a period of 5 to 90 days. We suggest the ways to modify existing HbA1c biosensors, leading to simple, reliable, and economical sensors ideally suited for point-of-care treatment.

An amperometric hemoglobin A1c biosensor based on immobilization of fructosyl amino acid oxidase onto zinc oxide nanoparticles-polypyrrole film.

Measurement of hemoglobin A1c (HbA1c, glycated hemoglobin) level in blood provides the long-term glucose level in diabetic patients without the influence of short-term fluctuations. The existing methods for HbA1c determination, including biosensors, suffer from insufficient sensitivity, detection limit, response time, and storage stability. These problems were overcome in the current biosensor. A method is described for construction of an amperometric HbA1c biosensor by immobilizing a fructosyl amino acid oxidase (FAO) onto zinc oxide nanoparticles/polypyrrole (ZnONPs/PPy) hybrid film deposited onto gold (Au) electrode and using it as working electrode, Ag/AgCl as reference electrode, and platinum (Pt) as auxiliary electrode. The whole blood samples were hemolyzed and digested by protease before measuring their HbA1c level by the biosensor. The enzyme electrode detected fructosyl valine (FV) as low as 50µM at a signal-to-noise ratio of 3 within 2s at +0.27V versus Ag/AgCl, pH7.0, and 35°C with a linear working range of 0.1 to 3.0mM for FV and sensitivity of 38.42µAmM(-1). The electrode showed only a 30% loss of its initial response over a period of 160days when stored at 4°C. The biosensor measured HbA1c in whole blood of apparently healthy individuals and diabetic patients and found it to be in the ranges of 4.0% to 5.6% and 5.7% to 12.0%, respectively.

An amperometric biosensor based on laccase immobilized onto Fe3O4NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract.

A method is described for the construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase onto iron oxide nanoparticles (Fe3O4NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto a gold (Au) electrode. The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 6.0 (0.1 M sodium acetate buffer) and 35°C, when operated at 0.3 V vs. Ag/AgCl. Linear range, detection limit were 0.1-10 µM (lower concentration range) and 10-500 µM (higher concentration range), and 0.03 µM respectively. The sensor measured total phenolic content in tea leaves extract. The enzyme electrode lost 25% of its initial activity after its 150 uses over a period of 4 months, when stored at 4°C.

Amperometric determination of total phenolic content in wine by laccase immobilized onto silver nanoparticles/zinc oxide nanoparticles modified gold electrode.

A method is described for construction of a highly sensitive amperometric biosensor for measurement of total phenolic compounds in wine by immobilizing laccase covalently onto nanocomposite of silver nanoparticles (AgNPs)/zinc oxide nanoparticles (ZnONPs) electrochemically deposited onto gold (Au) electrode. Scanning electron microscopy, X-ray diffraction, and electrochemical impedance spectroscopy were applied for characterization of the surface morphology of the modified electrode, and cyclic voltammetry was used to investigate the electrochemical properties of the proposed electrode toward the oxidation of guaiacol. The linearity between the oxidation current and the guaiacol concentration was obtained in a range of 0.1 to 500µM with a detection limit of 0.05µM (signal-to-noise ratio (S/N)=3) and sensitivity of 0.71µAµM(-1)cm(-2). The electrode showed increased oxidation and reduced reduction current with the deposition of AgNPs/ZnONPs on it. R(CT) values of ZnONPs/Au, AgNPs/ZnONPs/Au, and laccase/AgNPs/ZnONPs/Au electrode were 220, 175, and 380Ω, respectively. The biosensor showed an optimal response within 8s at pH 6.0 (0.1M acetate buffer) and 35°C when operated at 0.22V against Ag/AgCl. Analytical recovery of added guaiacol was 98%. The method showed a good correlation (r=0.99) with the standard spectrophotometric method, with the regression equation being y=1.0053x-3.5541. The biosensor lost 25% of its initial activity after 200 uses over 5months.

An electrochemical sulfite biosensor based on gold coated magnetic nanoparticles modified gold electrode.

A sulfite oxidase (SO(X)) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto carboxylated gold coated magnetic nanoparticles (Fe(3)O(4)@GNPs) electrodeposited onto the surface of a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC)-N-hydroxy succinimide (NHS) chemistry. An amperometric sulfite biosensor was fabricated using SO(X)/Fe(3)O(4)@GNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode. The working electrode was characterized by Fourier Transform Infrared (FTIR) Spectroscopy, Cyclic Voltammetry (CV), Scanning Electron Microscopy (SEM) and Electrochemical Impedance Spectroscopy (EIS) before and after immobilization of SO(X). The biosensor showed optimum response within 2s when operated at 0.2V (vs. Ag/AgCl) in 0.1 M Tris-HCl buffer, pH 8.5 and at 35 °C. Linear range and detection limit were 0.50-1000 µM and 0.15 µM (S/N=3) respectively. Biosensor was evaluated with 96.46% recovery of added sulfite in red wine and 1.7% and 3.3% within and between batch coefficients of variation respectively. Biosensor measured sulfite level in red and white wines. There was good correlation (r=0.99) between red wines sulfite value by standard DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) method and the present method. Enzyme electrode was used 300 times over a period of 4 months, when stored at 4 °C. Biosensor has advantages over earlier biosensors that it has excellent electrocatalysis towards sulfite, lower detection limit, higher storage stability and no interference by ascorbate, cysteine, fructose and ethanol.

Construction of amperometric uric acid biosensor based on uricase immobilized on PBNPs/cMWCNT/PANI/Au composite.

A chitosan-glutaraldehyde crosslinked uricase was immobilized onto Prussian blue nanoparticles (PBNPs) absorbed onto carboxylated multiwalled carbon nanotube (c-MWCNT) and polyaniline (PANI) layer, electrochemically deposited on the surface of Au electrode. The nanohybrid-uricase electrode was characterized by scanning electron microscopic (SEM), Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry. An amperometric uric acid biosensor was fabricated using uricase/c-MWCNT/PBNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The biosensor showed optimum response within 4s at pH 7.5 and 40°C, when operated at 0.4V vs. Ag/AgCl. The linear working range for uric acid was 0.005-0.8 mM, with a detection limit of 5 µM. The sensor was evaluated with 96% recovery of added uric acid in sera and 4.6 and 5.4% within and between batch of coefficient of variation respectively and a good correlation (r=0.99) with standard enzymic colorimetric method. This sensor measured uric acid in real serum samples. The sensor lost only 37% of its initial activity after its 400 uses over a period of 7 months, when stored at 4°C.

Development of an amperometric polyphenol biosensor based on fungal laccase immobilized on nitrocellulose membrane.

A method is described for construction of an amperometric polyphenol biosensor employing nitrocellulose membrane-bound laccase purified from cell-free extract of Ganoderma lucidum onto a Pt electrode. The biosensor showed optimum response within 10s, at 0.4 V in 0.1M acetate buffer, pH 6.0, and 35°C. Detection limit of the biosensor was 3.0 × 10(-8)M. Analytical recovery of added guaiacol was 97.00%. Within batch and between batch coefficients of variation were <0.97% and <1.26%, respectively. The sensor measured total phenolic content in fruit juices and alcoholic beverages. The enzyme electrode was used 100 times over 4 months, when stored at 4°C.

An amperometric biosensor based on laccase immobilized onto MnO2NPs/cMWCNT/PANI modified Au electrode.

A method is described for construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase (Lac) onto manganese dioxide nanoparticles (MnO(2)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/PANI composite electrodeposited onto a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response at pH 5.5 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3 V vs. Ag/AgCl. Linear range, response time, detection limit were 0.1-10 µM (lower concentration range) and 10-500 µM (higher concentration range), 4s and 0.04 µM, respectively. Biosensor measured total phenolic content in tea leaves extract. The enzyme electrode was used 150 times over a period of 5 months.

Fabrication of polyphenol biosensor based on laccase immobilized on copper nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode.

A high-performance amperometric polyphenol biosensor was developed, based on covalent immobilization of Ganoderma sp. laccase onto copper nanoparticles (CuNP's)/chitosan (CHIT)/carboxylated multiwalled carbon nanotube (cMWCNT)/polyaniline (PANI)-modified gold (Au) electrode. The CuNP's and cMWCNT had a synergistic electrocatalytic effect in the matrix of CHIT. The biosensor showed optimum response at pH 6.0 (0.1 M acetate buffer) and 35°C, when operated at 50 mVs(-1). The biosensor exhibited excellent sensitivity (the detection limit was down to 0.156 µM for guaiacol), fast response time (less than 4s) and wide linear range (from 1 to 500 µM). Analytical recovery of added guaiacol was 96.40-98.46%. Within batch and between batch coefficients of variation were <2.6% and <5.3%, respectively. The enzyme electrode was used 300 times over a period of 7 months, when stored at 4°C.

Development of an amperometric sulfite biosensor based on a gold nanoparticles/chitosan/multiwalled carbon nanotubes/polyaniline-modified gold electrode.

A sulfite oxidase (SOx) purified from leaves of Syzygium cumini (Jamun) was immobilized covalently onto a gold nanoparticles (AuNPs)/chitosan (CHIT)/carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite that was electrodeposited onto the surface of a gold (Au) electrode. A novel and highly sensitive sulfite biosensor was developed that used this enzyme electrode (SOx/AuNPs/CHIT/cMWCNT/PANI/Au) as the working electrode, Ag/AgCl as the standard electrode, and Pt wire as the auxiliary electrode. The modified electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS) before and after the immobilization of the SOx. The sensor produced its optimum response within 3 s when operated at 50 mVs(-1) in 0.1 M phosphate buffer, pH 7.0, and at 35 °C. The linear range and detection limit of the sensor were 0.75-400 µM and 0.5 µM (S/N = 3), respectively. The biosensor was employed to determine sulfite levels in fruit juices and alcoholic beverages. The enzyme electrode was used 300 times over a period of three months when stored at 4 °C.

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode.

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35°C when operated at a scan rate of 50 mV s(-1). Linear range, response time, and detection limit were 0.1-500 µM, 6 s, and 0.1 µM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4°C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.

An electrochemical biosensor for fructosyl valine for glycosylated hemoglobin detection based on core-shell magnetic bionanoparticles modified gold electrode.

A high-performance amperometric fructosyl valine (FV) biosensor was developed, based on immobilization of fructosyl amino-acid oxidase (FAO) on core-shell magnetic bionanoparticles modified gold electrode. Chitosan was used to introduce amino groups onto the surface of core-shell magnetic bionanoparticles (MNPs). With FAO as an enzyme model, a new fructosyl valine biosensor was fabricated. The biosensor showed optimum response, when operated at 50 mVs(-1) in 0.1M potassium phosphate buffer, pH 7.5 and 35°C. The biosensor exhibited excellent sensitivity [the detection limit is down to 0.1mM for FV], fast response time (less than 4s), wide linear range (from 0 to 2mM). Analytical recovery of added FV was 95.00-98.50%. Within batch and between batch coefficients of variation were <2.58% and <5.63%, respectively. The enzyme electrode was used 250 times over 3 months, when stored at 4°C.

An amperometric polyphenol biosensor based on laccase immobilized on epoxy resin membrane.

A polyphenol biosensor employing epoxy resin membrane bound Ganoderma sp. laccase has been developed. The biosensor showed optimum response within 30 s, when operated at 0.4 V in 0.1 M acetate buffer, pH 6.0 and 35 °C. Detection limit of the biosensor was 3.0 x 10-7 M. Analytical recovery of added guaiacol was 96.66%. Within batch and between batch coefficients of variation were <1.35% and <2.97% respectively. The biosensor was employed for amperometric determination of polyphenols in fruit juices and alcoholic beverages. The enzyme electrode was used 200 times over a period of 10 months, when stored at 4 °C.