Label-free reflectometric interference microchip biosensor based on nanoporous alumina for detection of circulating tumour cells

In this report, a label-free reflectometric interference spectroscopy (RIfS) based microchip biosensor for the detection of circulating tumour cells (CTCs) is demonstrated. Highly ordered nanoporous anodic aluminium oxide (AAO) fabricated by electrochemical anodization of aluminium foil was used as the RIfS sensing platform. Biotinylated anti-EpCAM antibody that specifically binds to human cancer cells of epithelial origin such as pancreatic cancer cells (PANC-1) was covalently attached to the AAO surface through multiple surface functionalization steps. Whole blood or phosphate buffer saline spiked with low numbers of pancreatic cancer cells were successfully detected by specially designed microfluidic device incorporating an AAO RIfS sensor, without labour intensive fluorescence labelling and/or pre-enhancement process. Our results show that the developed device is capable of selectively detecting of cancer cells, within a concentrations range of 1000-100,000 cells/mL, with a detection limit of <1000 cells/mL, a response time of <5 min and sample volume of 50 μL of. The presented RIfS method shows considerable promise for translation to a rapid and cost-effective point-of-care diagnostic device for the detection of CTCs in patients with metastatic cancer.     

Sarcosine Oxidase Involved in Creatinine Degradation in Alcaligenes denitrificans subsp. denitrificans J9 and Arthrobacter spp. J5 and J11

Three microorganisms that degrade creatinine and contain sarcosine oxidase were isolated from soil and identified to be Alcaligenes denitrificans subsp. denitrificans J9 and Arthrobacter spp. J5 and J11. The three soil isolates degraded creatinine only via creatine by inducibly formed creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase when cultivated with creatinine as the main nitrogen source. Sarcosine dehydrogenase, creatinine deiminase, and N-carbamoylsarcosine amidohydrolase were not induced by creatinine. Other microorganisms that degrade creatinine all contain sarcosine dehydrogenase as the enzyme for sarcosine oxidation, so these isolates seem to be unique in having sarcosine oxidase involved in their processes of creatinine degradation. Sarcosine oxidase was purified from A. denitrificans subsp. denitrificans J9 and partially characterized.     

Purification and Properties of Sarcosine Oxidase from Cylindrocarpon didymum M–1

Sarcosine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing choline as the carbon source. The molecular weight of the enzyme was estimated to be 45,000 by gel filtration method and 48,000 by the sodium dodecylsulfate disc gel electrophoresis method. The enzyme exhibited an absorption spectrum with maxima at 277 and 450 run and shoulders at 370 and 470 nm. The anaerobic addition of sarcosine to the enzyme resulted in the disappearance of the peak at 450 nm. The enzyme contained one mol of covalently bound FAD per mol of enzyme. Enzyme activity was inhibited by Ag⁺, Cu²⁺, Hg²⁺, p-chloromercuribenzoate and iodoacetate. The enzyme oxidized sarcosine but was inert toward choline, betaine, dimethylglycine and N-methyl amino acids. Km and V_max values for sarcosine were 1.8 ihm and 26.2 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Sarcosine+O₂+H₂O→glycine +formaldehyde+H₂O₂.     

Eco‐friendly synthesis of gelatin‐capped bimetallic Au–Ag nanoparticles for chemiluminescence detection of anticancer raloxifene hydrochloride

This study described the utility of green analytical chemistry in the synthesis of gelatin‐capped silver, gold and bimetallic gold–silver nanoparticles (NPs). The preparation of nanoparticles was based on the reaction of silver nitrate or chlorauric acid with a 1.0 wt% aqueous gelatin solution at 50°C. The gelatin‐capped silver, gold and bimetallic NPs were characterized using transmission electron microscopy, UV–vis, X‐ray diffraction and Fourier transform infrared spectroscopy, and were used to enhance a sensitive sequential injection chemiluminescence luminol–potassium ferricyanide system for determination of the anticancer drug raloxifene hydrochloride. The developed method is eco‐friendly and sensitive for chemiluminescence detection of the selected drug in its bulk powder, pharmaceutical injections and biosamples. After optimizing the conditions, a linear relationship in the range of 1.0 × 10^–9 to 1.0 × 10^–1 mol/L was obtained with a limit of detection of 5.0 × 10^–10 mol/L and a limit of quantification of 1.0 × 10^‐9 mol/L. Statistical treatment and method validation were performed based on ICH guidelines. Copyright © 2016 John Wiley & Sons, Ltd.     

Thermal stability of human haemoglobin in the presence of sarcosine and sorbitol

Sarcosine and sorbitol at 10–30% (w/w) stabilized haemoglobin from human erythrocytes to thermal denaturation. At 65?°C, the protein's half life was increased from 0.85 to 50 min in 30% sarcosine and to 24 min in 30% sorbitol. A kinetic analysis based on the Lumry-Eyring mechanism of inactivation showed that the denaturation process can be described by a second-order rate expression with an apparent activation energy ranging from 56 to 87 kcal mol^?1.     

Novel polyhedral gold nanoparticles: green synthesis,optimization and characterization by environmental isolate of Acinetobacter sp. SW30

Gold nanoparticles have enormous applications in cancer treatment, drug delivery and nanobiosensor due to their biocompatibility. Biological route of synthesis of metal nanoparticles are cost effective and eco-friendly. Acinetobacter sp. SW 30 isolated from activated sewage sludge produced cell bound as well as intracellular gold nanoparticles when challenged with HAuCl₄ salt solution. We first time report the optimization of various physiological parameters such as age of culture, cell density and physicochemical parameters viz HAuCl₄ concentration, temperature and pH which influence the synthesis of gold nanoparticles. Gold nanoparticles thus produced were characterized by various analytical techniques viz. UV–Visible spectroscopy, X-ray diffraction, cyclic voltammetry, transmission electron microscopy, selected area electron diffraction, high resolution transmission electron microscopy, environmental scanning electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy and dynamic light scattering. Polyhedral gold nanoparticles of size 20 ± 10 nm were synthesized by 24 h grown culture of cell density 2.4 × 10⁹ cfu/ml at 50 °C and pH 9 in 0.5 mM HAuCl₄. It was found that most of the gold nanoparticles were released into solution from bacterial cell surface of Acinetobacter sp. at pH 9 and 50 °C.     

Determination of fluoxetine in pharmaceutical and biological samples based on the silver nanoparticle enhanced fluorescence of fluoxetine–terbium complex

In this study, a simple and sensitive spectrofluorimetric method is presented for the determination of fluoxetine based on the enhancing effect of silver nanoparticles (AgNPs) on the terbium–fluoxetine fluorescence emission. The AgNPs were prepared by a simple reduction method and characterized by UV–Vis spectroscopy and transmission electron microscopy. It was indicated that these AgNPs have a remarkable amplifying effect on the terbium‐sensitized fluorescence of fluoxetine. The effects of various parameters such as AgNP and Tb³⁺ concentration and the pH of the media were investigated. Under obtained optimal conditions, the fluorescence intensity of the terbium–fluoxetine–AgNP system was enhanced linearly by increasing the concentration of fluoxetine in the range of 0.008 to 19 mg/L. The limit of detection (b + 3s) was 8.3 × 10^‐4 mg/L. The interference effects of common species found in real samples were also studied. The method had good linearity, recovery, reproducibility and sensitivity, and was satisfactorily applied for the determination of fluoxetine in tablet formulations, human urine and plasma samples. Copyright © 2016 John Wiley & Sons, Ltd.     

Reductive cleavage of sarcosine and betaine by Eubacterium acidaminophilum via enzyme systems different from glycine reductase

The obligate anaerobe Eubacterium acidaminophilum metabolized the glycine derivatives sarcosine (N-monomethyl glycine) and betaine (N-trimethyl glycine) only by reduction in a reaction analogous to glycine reductase. Using formate as electron donor, sarcosine and betaine were stoichiometrically reduced to acetate and methylamine or trimethylamine, respectively. The N-methyl groups of the cosubstrates or of the amines produced were not transformed to CO₂ or acetate. Under optimum conditions (formate/acceptor ratio of 1 to 1.2, 34°C, pH 7.3) the doubling times were 4.2 h on formate/sarcosine and 3.6 h on formate/betaine. The molar growth yields were 8.15 and 8.5 g dry cell mass per mol sarcosine and betaine, respectively. The assays for sarcosine reductase and betaine reductase were optimized in cell extracts; NADPH was preferred as physiological electron donor compared to NADH, dithioerythritol was used as artificial donor; no requirements for AMP and ADP could be detected. Growth experiments mostly revealed diauxic substrate utilization pattern using different combinations of glycine, sarcosine, and betaine (plus formate) and inocula from different precultures. Glycine was always utilized first, what coincided with the presence of glycine reductase activity under all growth conditions except for serine as substrate. Sarcosine reductase and betaine reductase were only induced when E. acidaminophilum was grown on sarcosine and betaine, respectively. Creatine was metabolized via sarcosine. [⁷⁵Se]-selenite labeling revealed about the same pattern of predominant labeled proteins in glycine-, sarcosine-, and betaine-grown cells.Abbreviations DTE dithioerythritol - TES N-Tris (hydroxymethyl) methyl-2-amino-ethane sulfonic acid     

Biological synthesis of platinum nanoparticles using Diopyros kaki leaf extract

The leaf extract of Diopyros kaki was used as a reducing agent in the ecofriendly extracellular synthesis of platinum nanoparticles from an aqueous H₂PtCl₆·6H₂O solution. A greater than 90% conversion of platinum ions to nanoparticles was achieved with a reaction temperature of 95°C and a leaf broth concentration of >10%. A variety of methods was used to characterize the platinum nanoparticles synthesized: inductively coupled plasma spectrometry, transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). The average particle size ranged from 2 to 12 nm depending on the reaction temperature and concentrations of the leaf broth and PtCl₆ ²⁻. FTIR analysis suggests that platinum nanoparticle synthesis using Diopyros kaki is not an enzyme-mediated process. This is the first report of platinum nanoparticle synthesis using a plant extract.     

Molecularly imprinted electrochemiluminescence sensor based on ZIF-8 doped with CdSe quantum dots for the detection of trace estriol

A composite of the metal–organic framework compound ZIF-8 doped with CdSe quantum dots (QDs) with sensitive and stable luminescence was synthesized, and a molecularly imprinted electrochemiluminescence (ECL) sensor was constructed based on this composite. The ZIF-8@CdSe molecularly imprinted ECL sensor combines the high sensitivity of ECL and the high selectivity of molecular imprinting to realize the sensitive and specific detection of estriol. CdSe QDs and gold nanoparticles were encapsulated within ZIF-8 to obtain the ZIF-8@CdSe QDs/GNP (ZIF@CdSe/GNP) composite. Subsequently, the GNPs were further loaded on the surface of this composite to obtain the GNP/ZIF@CdSe/GNP composite. l -Cysteine was used to immobilize the GNP/ZIF@CdSe/GNP composite on the surface of a gold electrode to obtain the GNP/ZIF@CdSe/GNP-modified gold electrode. A molecularly imprinted polymer (MIP) film was prepared on the surface of the modified electrode by electropolymerization with o-phenylenediamine as the functional monomer and estriol as the template molecule. After elution, estriol could be specifically recognized by the cavities. The readsorption of estriol by the MIP can prevent the coreactant from reaching the electrode surface through the cavities, thereby weakening ECL. A good linear relationship existed between the ∆ECL and lg C of estriol concentrations of 1 × 10⁻¹⁴ to 1 × 10⁻⁹ mol·L⁻¹. The detection limit was as low as 8.9 × 10⁻¹⁶ mol·L⁻¹. The sensor was applied in the determination of estriol in serum samples with a recovery of 97.0–102%.     

A sensitive fluorescent nanosensor for chloramphenicol based on molecularly imprinted polymer‐capped CdTe quantum dots

A novel fluorescent nanosensor using molecularly imprinted silica nanospheres embedded CdTe quantum dots (CdTe@SiO₂@MIP) was developed for detection and quantification of chloramphenicol (CAP). The imprinted sensor was prepared by synthesis of molecularly imprinting polymer (MIP) on the hydrophilic CdTe quantum dots via reverse microemulsion method using small amounts of solvents. The resulting CdTe@SiO₂@MIP nanoparticles were characterized by fluorescence, UV–vis absorption and FT‐IR spectroscopy and transmission electron microscopy. They preserved 48% of fluorescence quantum yield of the parent quantum dots. CAP remarkably quenched the fluorescence of prepared CdTe@SiO₂@MIP, probably via electron transfer mechanism. Under the optimal conditions, the relative fluorescence intensity of CdTe@SiO₂@MIP decreased with increasing CAP by a Stern–Volmer type equation in the concentration range of 40–500 µg L^–1. The corresponding detection limit was 5.0 µg L^–1. The intra‐day and inter‐day values for the precision of the proposed method were all <4%. The developed sensor had a good selectivity and was applied to determine CAP in spiked human and bovine serum and milk samples with satisfactory results. Copyright © 2015 John Wiley & Sons, Ltd.     

Chiral determination of cinchonine using an electrochemiluminescent sensor with molecularly imprinted membrane on the surfaces of magnetic particles

A novel molecular imprinting electrochemiluminescence sensor for detecting chiral cinchonine molecules was developed with a molecularly imprinted polymer membrane on the surfaces of magnetic microspheres. Fe₃O₄@Au nanoparticles modified with 6‐mercapto‐beta‐cyclodextrin were used as a carrier, cinchonine as a template molecule, methacrylic acid as a functional monomer and N ,N ′‐methylenebisacrylamide as a cross‐linking agent. Cinchonine was specifically recognized by the 6‐mercapto‐beta‐cyclodextrin functional molecularly imprinted polymer and detected based on enhancement of the electrochemiluminescence intensity caused by the reaction of tertiary amino structures of cinchonine molecules with Ru(bpy)₃²⁺. Cinchonine concentrations of 1 × 10^?10 to 4 × 10^?7 mol/L showed a good linear relationship with changes of the electrochemiluminescence intensity, and the detection limit of the sensor was 3.13 × 10^?11 mol/L. The sensor has high sensitivity and selectivity, and is easy to renew. It was designed for detecting serum samples, with recovery rates of 98.2% to 107.6%.     

A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces.

Label-free heterogeneous phase detection critically depends on the properties of the interfacial layer. We have obtained high-density monomolecular poly(ethylene glycol) (PEG) layers by solvent-free coupling of homo-bifunctional PEGs (2,000 g/mol) at 75 degrees C to silica surfaces silanized with glycidyloxipropyltrimethoxysilane (GOPTS). Characterization by ellipsometry and contact angles revealed that PEG layers up to 3.4 ng/mm2 with low roughness and flexibility were obtained. Specific and non-specific binding at these PEG surfaces was monitored by reflectometric interference spectroscopy (RIfS). No significant non-specific adsorption upon incubation of 1 mg/ml ovalbumin was detectable (< 10 pg/mm2), and 150 pg/mm2 upon incubation of 10% calf serum, less than 10% of the amount adsorbed to the solely silanized surfaces. The terminal functional groups of the PEG layers were utilized to couple ligands and a protein. Specific protein interaction with these immobilized compounds was detected with saturation loadings in the range of protein monolayers (2-4 ng/mm2). The excellent functional properties, the high stability of the layers, the generic and practical coupling procedure and the versatility for immobilizing compounds of very different functionality make these PEG layers very attractive for application in label-free detection with silica or metal-oxide based transducers.     

Mutations in the sarcosine dehydrogenase gene in patients with sarcosinemia

Sarcosinemia is an autosomal recessive metabolic trait manifested by relatively high concentrations of sarcosine in blood and urine. Sarcosine is a key intermediate in 1-carbon metabolism and under normal circumstances is converted to glycine by the enzyme sarcosine dehydrogenase. We encountered six families from two different descents (French and Arab), each with at least one individual with elevated levels of sarcosine in blood and urine. Using the “candidate gene approach” we sequenced the gene encoding sarcosine dehydrogenase (SARDH), which plays an important role in the conversion of sarcosine to glycine, and found four different mutations (P287L, V71F, R723X, R514X) in three patients. In an additional patient, we found a uniparental disomy in the region of SARDH gene. In two other patients, we did not find any mutations in this gene. We have shown for the first time that mutations in the SARDH gene are associated with sarcosinemia. In addition, our results indicate that other genes are most probably involved in the pathogenesis of this condition.     

CdS nanoparticles‐ enhanced chemiluminescence and determination of baicalin in pharmaceutical preparations

CdS nanoparticles (CdS NPs) of different sizes were synthesized by the citrate reduction method. It was found that CdS NPs could enhance the chemiluminescence (CL) of the luminol‐potassium ferricyanide system and baicalin could inhibit CdS NPs‐enhanced luminol‐potassium ferricyanide CL signals in alkaline solution. Based on this inhibition, a flow‐injection CL method was established for determination of baicalin in pharmaceutical preparations and human urine samples. Under optimized conditions, the linear range for determination of baicalin was 5.0 x 10^?6 to 1.0 x 10^?3 g/L. The detection limit at a signal‐to‐noise ratio of 3 was 1.7 x 10 ^?6 g/L. CL spectra, UV‐visible spectra and transmission electron microscopy (TEM) were used to investigate the CL mechanism. The method described is simple, selective and obviates the need of extensive sample pretreatment. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of nano‐sized hydrogen phosphate‐imprinted polymer in acetonitrile/water mixture and its use as a recognition element of hydrogen phosphate selective all‐solid state potentiometric electrode

Herein, a new recipe is introduced for the preparation of hydrogen phosphate ion‐imprinted polymer nanoparticles (nano‐IIP) in acetonitrile/water (63.5:36.5) using phosphoric acid as the template. The nano‐IIP obtained was used as the recognition element of a carbon paste potentiometric sensor. The IIP electrode showed a Nernstian response to hydrogen phosphate anion; whereas, the non‐imprinted polymer (NIP)‐based electrode had no considerable sensitivity to the anion. The presence of both methacrylic acid and vinyl pyridine in the IIP structure, as well as optimization of the functional monomers‐template proportion, was found to be important to observe the sensing capability of the IIP electrode. The nano‐IIP electrode showed a dynamic linear range of 1 × 10^?5‐1 × 10^?1 mol L‐1, Nernstian slope of 30.6 ± (0.5) mV decade ^?1, response time of 25 seconds, and detection limit of 4.0 × 10^?6 mol L^?1. The utility of the electrodes was checked by potentiometric titration of hydrogen phosphate with La³⁺ solution.     

Surface plasmon resonance sensor for femtomolar detection of testosterone with water-compatible macroporous molecularly imprinted film

A novel water-compatible macroporous molecularly imprinted film (MIF) has been developed for rapid, sensitive, and label-free detection of small molecule testosterone in urine. The MIF was synthesized by photo copolymerization of monomers (methacrylic acid [MAA] and 2-hydroxyethyl methacrylate [HEMA]), cross-linker (ethylene glycol dimethacrylate, EGDMA), and polystyrene nanoparticles (PS NPs) in combination with template testosterone molecules. The PS NPs and template molecules were subsequently removed to form an MIF with macroporous structures and the specific recognition sites of testosterone. Incubation of artificial urine and human urine on the MIF and the non-imprinted film (NIF), respectively, indicated undetectable nonspecific adsorption. Accordingly, the MIF was applied on a surface plasmon resonance (SPR) sensor for the detection of testosterone in phosphate-buffered saline (PBS) and artificial urine with a limit of detection (LOD) down to 10⁻¹⁵ g/ml. To the best of our knowledge, the LOD is considered as one of the lowest among the SPR sensors for the detection of small molecules. The control experiments performed with analogue molecules such as progesterone and estradiol demonstrated the good selectivity of this MIF for sensing testosterone. Furthermore, this MIF-based SPR sensor shows high stability and reproducibility over 8 months of storage at room temperature, which is more robust than protein-based biosensors.     

Cloning of dimethylglycine dehydrogenase and a new human inborn error of metabolism, dimethylglycine dehydrogenase deficiency

Dimethylglycine dehydrogenase (DMGDH) (E.C. number 1.5.99.2) is a mitochondrial matrix enzyme involved in the metabolism of choline, converting dimethylglycine to sarcosine. Sarcosine is then transformed to glycine by sarcosine dehydrogenase (E.C. number 1.5.99.1). Both enzymes use flavin adenine dinucleotide and folate in their reaction mechanisms. We have identified a 38-year-old man who has a lifelong condition of fishlike body odor and chronic muscle fatigue, accompanied by elevated levels of the muscle form of creatine kinase in serum. Biochemical analysis of the patient's serum and urine, using (1)H-nuclear magnetic resonance NMR spectroscopy, revealed that his levels of dimethylglycine were much higher than control values. The cDNA and the genomic DNA for human DMGDH (hDMGDH) were then cloned, and a homozygous A-->G substitution (326 A-->G) was identified in both the cDNA and genomic DNA of the patient. This mutation changes a His to an Arg (H109R). Expression analysis of the mutant cDNA indicates that this mutation inactivates the enzyme. We therefore confirm that the patient described here represents the first reported case of a new inborn error of metabolism, DMGDH deficiency.     

Determination of phenformin hydrochloride using molecular imprinting technology coupled with flow-injection chemiluminescence

A novel method was developed using molecular imprinting technology (MIT) coupled with flow‐injection chemiluminescence (FI‐CL) for highly sensitive detection of phenformin hydrochloride (PH). The phenformin imprinted polymer was synthesized with methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as a cross‐linker. Newly synthesized molecularly imprinted polymer (MIP) particles were packed into a column as a selective recognition element for determination of PH. A CL method for the determination of PH was developed based on the CL reaction of PH with N‐bromosuccinimide sensitized by eosin Y in basic media. The optimization of detection conditions was investigated. The CL intensity responded linearly to the concentration of PH in the range 0.09–2.0 µg/mL, with a correlation coefficient of 0.9920. The detection limit was 0.031 µg/mL. The relative standard deviation for the determination of 1.0 µg/mL PH solution was 1.0% (n = 11). The method was applied to the determination of PH in urine samples, with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and β subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; β subunit was a tetramer that had sarcosine oxidase and l-proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αβ)₄ form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and β subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αβ)₄ complex may separately exist as a function enzyme in different conditions.