A Selective Determination of Hypoxanthine,Xanthine and Inosine by Reversed-Phase Liquid Chromatography Coupled with Enzyme Reactors

Abstract

A Selective and sensitive assay of hypoxyanthine, xanthine and inosine by reversed-phase liquid chromatography coupled with immobilized enzyme reactors is described. The flourometric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of hypoxanthine, xanthine and inosine, which were oxidized to hydrogen peroxide in the presence of the immobilized enzymes (purine nucleoside phosphorylase and/or xanthine oxidase. The enzymes were immobilized the the intermolecular cross-linking method on controlled pore-glass. The method established was applied to serum and urine samples. The detection limits of hypoxanthine, xanthine and inosine were approximately 130, 300 and 650 pg per injection, respectively.     

Quantitation of Femtomolar‐Level Protein Biomarkers Using a Simple Microbubbling Digital Assay and Bright‐Field Smartphone Imaging

Quantitating ultra‐low concentrations of protein biomarkers is critical for early disease diagnosis and treatment. However, most current point‐of‐care (POC) assays are limited in sensitivity. Herein, we introduce an ultra‐sensitive and facile microbubbling assay for the quantification of protein biomarkers with a digital‐readout method that requires only a smartphone camera. We used machine learning to develop a smartphone application for automated image analysis to facilitate accurate and robust counting. Using this method, post‐prostatectomy surveillance of prostate specific antigen (PSA) can be achieved with a detection limit (LOD) of 2.1 fm (0.060 pg mL^?1), and early pregnancy detection using βhCG can be achieved with a of 0.034 mIU mL^?1 (2.84 pg mL^?1). This work provides the proof‐of‐principle of the microbubbling assay with a digital readout as an ultra‐sensitive technology with minimal requirement for power and accessories, facilitating future POC applications.     

Selection of a method for determination of activity of pectinolytic enzymes in berry fruit materials

The goal of the study was to find the most accurate and sensitive method for the determination of activity of pectinolytic enzymes in complex mixtures obtained from fruit materials such as raw cloudberry and raspberry juices. Several assay methods based on enzymatic reactions using viscometric, colourimetric, spectrophotometric, or pH-titration detection of the reaction products were tested. Problems with the application of the selected methods, such as very low detection signal or very large background signal, were observed. Among the tested methods, only a modified method based on the continuous recording of the released carboxyl groups titration allowed to assay the activity of exogenous pectin methylesterase with a good linearity, sensitivity, accuracy, and minimised the interference of other fruit components.     

Design and synthesis of chemiluminescent probes for the detection of cholinesterase activity

Acetylcholinesterase is one of the most widely used and studied enzymes. Not only does this enzyme regulate neurotransmission (and thus play a key role in neurodegenerative processes) but it is also a prime target for pest control agents and warfare agents. Above all, due to its particularly high turnover rate, acetylcholinesterase is among the most efficient reporter enzymes yet described (for use as enzymatic tracer in immunoassays, for instance). However, its activity is detected through a colorimetric reagent, the Ellman reagent, which displays low detection limits and is often subject to background perturbations. In the course of our search for a more sensitive detection assay, we describe here a first-generation 1,2-dioxetane chemiluminescent probe, based on chemically induced electron exchange luminescence, which has an approximately 10 times lower detection limit than the Ellman colorimetric assay (2.5 x 10(-19) mol for Electrophorus electricus AChE in its tetrameric form).     

pH Readout enhanced ELISA for point-of-care testing of cardiac troponin I

In this study, we described a pH ELISA using synthetic melanin nanoparticles (SMNPs) for the co-immobilization of glucose oxidase and second antibody as signal labels, portable pH meter as signal readout device for detecting biomarker of myocardial injury. This assay is easy-to-use, portable, sensitive and able to realize point-of-care testing (POCT), which was demonstrated the signifi cant promising in the early diagnosis and screening of acute myocardial infarction.     

Detection of endogenous salsolinol in neonatal rat tissue by a radioenzymatic--thin-layer chromatographic assay

A sensitive radioenzymaticc--thin-layer chromatographic assay for the quantitative analysis of the tetrahydroisoquinoline alkaloid, salsolinol, in plasma and neonatal rat tissue is described. The assay involves the enzymatic O-methylation of salsolinol by catechol-O-methyltransferase in presence of [3H]S-adenosylmethionine, and subsequent separation by thin-layer chromatography of the resultant [3H]O-methyl-salsolinol from the O-methylated derivatives of dopamine, epinephrine and norepinephrine. The method allows the detection of as little as 100 pg salsolinol per g tissue, and the accurate quantitation of as little as 100 pg/ml plasma and 500 pg/g tissue. This assay permitted the detection of trace amounts of endogenous salsolinol in neonatal rat tissue (less than 500 pg/g tissue).     

An Enzyme Coupling Procedure for the Determination of Lipoprotein Lipase Activity

Abstract

A novel, sensitive enzyme coupling procedure is described for the quantitative determination of both purified bovine milk lipoprotein lipase (LPL) and human post-heparin plasma lipolytic activity (PHLA). The primary assay is carried out with the aid of a triolein emulsion optimized as regards NaCl, bile salt and C II apolipoprotein concentration. The secondary reaction consists of the photometric measurement of a colored adduct formed following enzymatic oxidation of non esterified fatty acids. The use of radiolabels is thus eliminated. The precision of the assay was comparable to other competent methods (CVs obtained are between 2.1 and 9.5). The system described is sensitive enough for detecting post-heparin plasma lipolytic activity in both healthy humans and cancer patients.     

Quantitative turbidity assay for lipolytic enzymes in microtiter plates

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

Mass spectrometry assay as an alternative to the enzyme-linked immunosorbent assay test for biomarker quantitation in ecotoxicology: Application to vitellogenin in Crustacea (Gammarus fossarum)

Vitellogenin (Vg) is a widespread biomarker for measuring exposure to endocrine disruptors. Vg quantification is usually done by using the ELISA test (enzyme-linked immunosorbent assay). Since this test is specific to a target protein, it can rarely be used with other species due to low cross-reactivity across species. Therefore alternative analytical methods have to be considered as the development of a specific and sensitive ELISA test for each new target is time-consuming and may prove unsuccessful. This paper focuses on the development of a quantitative assay by liquid chromatography tandem mass spectrometry (LC-MS/MS) of vitelogenin in an invertebrate (Gammarus fossarum) for which no ELISA kit is available. The linearity of the method was within the concentration range 2.5–25,000 pg/mL and the limit of detection was estimated at 0.75 pg/mL of Vg. This method has been demonstrated to be an alternative to existing immunological methods for quantifying Vg in invertebrates due to its greater sensitivity, specificity and reproducibility (intra- and inter-assay below 15%). This assay was applied for Vg determination in female G. fossarum following exposure to a known endocrine disruptor, methyl farnesoate, in crustaceans. The availability of a quantitative G. fossarum LC-MS/MS assay should open the way for further studies to evaluate xenoestrogen effects in aquatic male G. fossarum.     

Simultaneous assay of hypoxanthine, xanthine and allopurinol by high-performance liquid chromatography and activation of immobilized xanthine oxidase as an enzyme reactor

A selective and sensitive assay of substrates (hypoxanthine, xanthine and allopurinol) of xanthine oxidase by reversed-phase liquid chromatography coupled with the use of immobilized enzyme reactors is described. These compounds were oxidized by immobilized xanthine oxidase and produced hydrogen peroxide, which was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. The detection limits of hypoxanthine, xanthine and allopurinol were approximately 50, 120 and 130 pg per injection, respectively. Immobilized xanthine oxidase inhibited by oxipurinol during the assay was reactivated by 2,6-dichlorophenolindophenol and could be used for a long period without a significant activity loss. These methods were applied to plasma and urine samples.     

Determination of Estradiol by Biotin‐Avidin‐Amplified Electrochemical Enzyme Immunoassay

Abstract

A simple, sensitive biotin‐avidin‐amplified electrochemical enzyme‐linked immunosorbent assay (ELISA) for the determination of estradiol (E₂) was proposed in this paper. The complex of biotinylated anti‐E₂ antibody and horseradish peroxidase‐labeled avidin (HRP‐avidin) were regarded as a probe in this system. The activity of labeled enzyme was measured with electrochemical methods using o‐phenylenediamine as substrate. Coupled with the plate‐coated antigen, indirect ELISA format using E₂‐ovalbumin, the electrochemical detection was performed for E₂ with the detection limit of 21.0 pg/ml, and the linear range of determination of 50.0–500.0 pg/ml. The proposed method has been used for the determination of E₂ in river water with satisfactory results. Compared with the traditionally spectrophotometric ELISA detection, this method shows greatly heightened sensitivity. The limit of detection improved by about two orders of magnitude, which is very suitable for the conditions with extremely low concentration of analyte or very small volumes of sample present.     

Ultrahigh sensitivity assays for human cardiac troponin I using TiO2 nanotube arrays

Rapid, highly sensitive troponin assays for the analysis of serum at the point-of-care are particularly desirable for the effective treatment of myocardial infarction (MYI). TiO(2) nanotube arrays constitute a low cost, high surface area, semiconducting architecture with great promise for biosensing applications due to their compatibility with multiple detection techniques. Using TiO(2) nanotube arrays functionalized with highly robust and ordered carboxyalkylphosphonic acid self-assembled monolayers, we have developed a simple and highly sensitive fluorescence immunoassay which can detect concentrations of human cardiac troponin I as low as 0.1 pg ml(-1) without the use of enzymatic amplification. Varying the morphological parameters of the nanotube arrays allows tuning the detection range over 6 orders of magnitude of the troponin concentration from 0.1 pg ml(-1)-100 ng ml(-1).     

Rapid and sensitive detection of cholera toxin using gold nanoparticle-based simple colorimetric and dynamic light scattering assay

Herein, a rapid and simple gold nanoparticle based colorimetric and dynamic light scattering (DLS) assay for the sensitive detection of cholera toxin has been developed. The developed assay is based on the distance dependent properties of gold nanoparticles which cause aggregation of antibody-conjugated gold nanoparticles in the presence of cholera toxin resulting discernible color change. This aggregation induced color change caused a red shift in the plasmon band of nanoparticles which was measured by UV–Vis spectroscopy. In addition, we employed DLS assay to monitor the extent of aggregation in the presence of different concentration of cholera toxin. Our assay can visually detect as low as 10 nM of cholera toxin which is lower than the previously reported colorimetric methods. The reported assay is very fast and showed an excellent specificity against other diarrhetic toxins. Moreover, we have demonstrated the feasibility of our method for cholera toxin detection in local lake water.     

p-nitrophenyllaurate: a substrate for the high-performance liquid chromatographic determination of lipase activity

Many assay procedures have been devised to measure lipolytic activity, but none is without problems. It is for this reason that new methods are still being proposed. In this work we have investigated the use of two esters of p-nitrophenol, the palmitic acid and lauric acid esters, as substrates for a highly sensitive high-performance liquid chromatographic method. Data on recovery, specific activity and reproducibility are reported only for the lauric ester, because the palmitic ester turned out to be a very poor substrate.     

Rapid and sensitive detection of lower respiratory tract infections by stuffer‐free multiplex ligation‐dependent probe amplification

Lower respiratory tract infection is one of the most common infectious diseases. However, conventional methods for detecting infectious pathogens are time‐consuming, and generally have a limited impact on early therapeutic decisions. We previously reported a rapid and sensitive method for detecting such pathogens using stuffer‐free multiplex ligation‐dependent probe amplification coupled with high‐resolution CE‐SSCP. In this study, we report an application of this method to the detection of respiratory pathogens. As originally configured, this method was capable of simultaneously detecting seven bacterial species responsible for lower respiratory tract infections, but its detection limit and assay time were insufficient to provide useful information for early therapeutic decisions. To improve sensitivity and shorten assay time, we added a target‐specific preamplification step, improving the detection limit from 50 pg of genomic DNA to 500 fg. We further decreased time requirements by optimizing the hybridization step, enabling the entire assay to be completed within 7 h while maintaining the same detection limit. Taken together, these improvements enable the rapid detection of infectious doses of pathogens (i.e. a few dozen cells), establishing the strong potential of the refined method, particularly for aiding early treatment decisions.     

Sub-nanogram per litre detection of the emerging contaminant progesterone with a fully automated immunosensor based on evanescent field techniques

Progesterone is reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity in experimental animals and it can be found in various surface waters which are partly used as drinking water resources. Therefore, immunoanalytical methods at a very low limit of detection (LOD) and a low limit of quantification (LOQ) are becoming more and more important for environmental analysis and especially for monitoring drinking water quality. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe a fully automated immunoassay for progesterone with a LOD in the sub-nanogram per litre range and a LOQ in the lower nanogram per litre range. In contrast to common analytical methods such as GC-MS or HPLC-MS, the biosensor used requires no sample pre-treatment and no sample pre-concentration. The basis of our sensitive assay is the antibody with a high affinity constant towards progesterone and the robust biosensor setup used.     

Application of high-performance liquid chromatography to the study of biogenic amine-related enzymes

The application of high-performance liquid chromatography to the study of biogenic amine-related enzymes is reviewed. Biogenic amines include catecholamines (dopamine, norepinephrine and epinephrine), indoleamines (serotonin and melatonin), imidazoleamines (histamine), polyamines (putrescine, spermidine and spermine) and acetylcholine. Three particular aspects are covered. The first aspect is the assay of enzyme activities of biogenic amine-related enzymes, such as tyrosine hydroxylase, tryptophan hydroxylase, aromatic L-amino acid decarboxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase. The introduction of highly sensitive assays of biogenic amines with electrochemical detection or fluorescence detection have made possible the non-isotopic assay of these activities, replacing the previously used radioisotopic methods. The second aspect is the purification of these enzymes. Since biogenic amine-synthesizing enzymes are generally unstable, rapid and efficient purification of these enzymes is very useful. The third aspect is the assay of biogenic amines (for example, acetylcholine and polyamines) using post-column derivatization with biogenic amine oxidases and electrochemical detection.     

Immunosensor for estrone with an equal limit of detection as common analytical methods

Immunoanalytical methods at a very low limit of detection (LOD) and a low limit of quantification (LOQ) are becoming more and more important for environmental analysis and especially for monitoring drinking water quality. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe a fully automated immunoassay for estrone with a LOD below 0.20 ng L^–1 and a LOQ below 1.40 ng L^–1. In contrast to common analytical methods such as GC-MS or HPLC-MS, the biosensor used requires no sample pre-treatment and pre-concentration. The basis of our sensitive assay is the antibody with a high affinity constant towards estrone. The very low amount of antibody per sample results in low validation parameters (LOD, LOQ, and IC50), but this assay for estrone represents the current device-related limitation of the River Analyser (RIANA).     

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.     

Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria

A tandem technique for the detection of very low levels E. coli within about 2 h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli β-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay (ELISA). The commercially available 4-methylumbelliferyl-β-D-galactoside and a newly prepared substrate, 4-methylcoumarin-3-propionate-7-O-β-D-galactoside, were used with an ELISA for 7-hydroxy-4-methylcoumarin to demonstrate the detection of low levels of E. coli. The 2 h test indicates that a few viable bacteria cells could be detected by the tandem procedure. The end point of the test is an ELISA with colorimetric measurement step. The novel approach retains the essential features of the microbial enzymatic detection procedures and provides a highly sensitive detection system that can be used for rapid screening or quantification of viable microbial cells in water samples. The tandem test is generic for commonly employed glycosidases and other marker enzymes for which 4-methylumbillerone substrates are available.