A homogeneous immunoassay for cyclic nucleotides based on chemiluminescence energy transfer.

A chemiluminescent derivative of cyclic AMP, aminobutylethylisoluminol succinyl cyclic AMP (ABEI-scAMP), was synthesized in order to develop a homogeneous immunoassay based on non-radiative energy transfer. ABEI-scAMP was chemiluminescent (5.1 X 10(18) luminescent counts X mol-1 at pH 13), pure (greater than 95%) stable and immunologically active. A conventional immunoassay was established using ABEI-scAMP and a solid-phase anti-(cyclic AMP) immunoglobulin G which could detect cyclic AMP at least down to 25fmol. A homogeneous immunoassay for cyclic AMP was established by measuring the shift in wavelength from 460 to 525nm which occurred when ABEI-scAMP was bound to fluorescein-labelled anti-(cyclic AMP) immunoglobulin G. The assay was at least as sensitive as the conventional radioimmunoassay using cyclic [3H]AMP and could measure cyclic AMP over the range 1-1000nM. The homogeneous chemiluminescent energy transfer assay was also able to quantify the association and dissociation of antibody-antigen complexes. Chemiluminescence energy transfer occurred between fluorescein-labelled antibodies and several other ABEI-labelled antigens (Mr values 314-150000) including progesterone, cyclic GMP, complement component C9 and immunoglobulin G. The results provide a homogeneous immunoassay capable of measuring free cyclic AMP under conditions likely to exist inside cells.     

Chemiluminescence energy transfer: a new technique applicable to the study of ligand--ligand interactions in living systems

Chemiluminescent molecules can be readily detected in the range 10(-15) to 10(-18) mol, and potentially at least down to 10(-20) mol reacting/s. The chemiluminescent compound aminobutylethylisoluminol (ABEI) and its isothiocyanate derivative have been synthesized. The ABEI was coupled to rabbit immunoglobulin and cyclic AMP. These labeled antigens were stable for at least 9 months and were used to establish chemiluminescent immunoassays. When these chemiluminescent-labeled antigens bound to their respective fluorescein-labeled antibodies, a wavelength shift towards the green was detected in the chemiluminescence. This was due to chemiluminescence energy transfer and used to establish an homogenous immunoassay which could measure these antigens in biological samples at least as sensitively as conventional radioimmunoassays.     

Biotin-4-fluorescein based fluorescence quenching assay for determination of biotin binding capacity of streptavidin conjugated quantum dots

The valency of quantum dot nanoparticles conjugated with biomolecules is closely related to their performance in cell tagging, tracking, and imaging experiments. Commercially available streptavidin conjugates (SAv QDs) are the most commonly used tool for preparing QD-biomolecule conjugates. The fluorescence quenching of biotin-4-fluorscein (B4F) provides a straightforward assay to quantify the number of biotin binding sites per SAv QD. The utility of this method was demonstrated by quantitatively characterizing the biotin binding capacity of commercially available amphiphilic poly(acrylic acid) Qdot ITK SAv conjugates and poly(ethylene glycol) modified Qdot PEG SAv conjugates with emission wavelengths of 525, 545, 565, 585, 605, 625, 655, 705, and 800 nm. Results showed that 5- to 30-fold more biotin binding sites are available on ITK SAv QDs compared to PEG SAv QDs of the same color with no systematic variation of biotin binding capacity with size.     

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

Determination of 7-methylguanine, N2,N2-dimethylguanosine, and pseudouridine in ultrafiltrated serum of healthy adults by high-performance liquid chromatography

7-Methylguanine (m7Gua), N2,N2-dimethylguanosine (m2(2)Guo), and pseudouridine (psi) are degradation products from RNA turnover and can be used as markers for the whole-body turnover of mRNA-cap, tRNA, and rRNA (in healthy individuals, urinary excretion of these catabolites follows a regular pattern; the relative molar ratio of psi:m7Gua:m2(2)Guo is approximately 100:19:6). HPLC methods were developed to measure serum concentrations of these RNA catabolites after deproteinization of the samples by ultrafiltration through microcollodion bags with a nominal exclusion Mr of 12,400. For healthy adults the following values (mean +/- SD) were found: psi, 2760 +/- 460 nmol/liter (n = 10); m7Gua, 129.7 +/- 24.0 nmol/liter (n = 13); m2(2)Guo, 31.0 +/- 3.7 nmol/liter (n = 9). The relative molar ratio of these substances in serum derived from our data is approximately 100:4.7:1.1. 7-Methylguanosine (m7Guo) added to serum is to a large extent converted to the corresponding free base, m7Gua, the form which is excreted in urine.     

Hydroperoxide-initiated chemiluminescence: an assay for oxidative stress in biopsies of heart, liver, and muscle.

Hydroperoxide-initiated chemiluminescence was standardized as a microassay to evaluate the occurrence of oxidative stress in human biopsies. Samples of 10 to 50 mg of rat liver or heart were homogenized, diluted in reaction medium, added with tert-butyl hydroperoxide, and assayed for chemiluminescence in a liquid scintillation counter in the out-of-coincidence mode. Optimal conditions for the assay were: 0.3 to 1.2 mg/mL of homogenate protein in 120 mM KCl, 30 mM phosphate buffer (pH 7.4), and 3 mM tert-butyl hydroperoxide at 30 degrees C. In these conditions, maximal chemiluminescence values were 550 +/- 30 and 1100 +/- 40 cps/mg protein, for liver and heart homogenates, respectively. Liver and heart homogenates were subjected to in vitro oxidative stresses such as supplementation with organic hydroperoxide or with enzymatic systems generating superoxide anion or hydrogen peroxide. Chemiluminescence was higher in the poststress samples than in the control ones. The ratio: poststress chemiluminescence/control chemiluminescence (B/A) was about 1.4 or higher for both tissues. Human heart biopsies were utilized to investigate the occurrence of oxidative stress after clinical situations associated to ischemia-reperfusion. B/A ratios were 2.1 +/- 0.4, 1.4 +/- 0.1, and 2.8 +/- 0.4 for human heart, liver, and skeletal muscle, respectively.     

Microbiological Assay for Organic Compounds in Seawater

A method for the quantitative identification of organic compounds in seawater has been developed. When auxotrophic mutants of Serratia marinorubra were incubated at 21 to 24 C for 72 hr with constant agitation, standard bioassay reference curves were obtained. Sodium glycerophosphate (400 mg per liter), ammonium dibasic citrate (5 g per liter), and glycerol (25 ml per liter) supplied the needed nutrients for maximal growth with a limited concentration of the required metabolite. Data are presented for the microbiological assay for biotin in waters of the Gulf of Mexico and adjacent bays. The range of sensitivity for the biotin mutant A101V is 5 to 12 mmug per liter in seawater, with a growth response from 2 to 16 mmug per liter of seawater. The possible ecological and chemical significance of biotin occurrence in spring-summer off-shore water is discussed.     

Eco-friendly nonconjugated polymer dots for chemiluminometric determination of 4-nitrophenol

Polymer dots (PDs) are a new family of quantum dots for which their behavior and potential applications have not yet been completely explored. In this study, nonconjugated PDs were synthesized using a simple pyrolysis method and used for the chemiluminescence (CL) assay of 4-nitrophenol (4-NP). PDs increased the CL signal of the Ce(IV)–Na₂SO₃ reaction 39-fold. Using the CL spectrum, it was concluded that the emission at 434 nm was generated by excited PDs (PDs*), which are produced by energy transfer from SO₂* to PDs. Our experiments showed that 4-NP enhanced the CL signal of the Ce(IV)–Na₂SO₃–PDs reaction. The mechanism of this effect was explored by obtaining CL, ultraviolet–visible, and Fourier transform infrared spectra. Due to the high sensitivity and selectivity of the CL system for 4-NP, a probe was designed to determine 4-NP in the linear range 1.0–500 nmol/L with a detection limit of 0.33 nmol/L. Different spiked real samples were successfully analyzed using this probe.     

The quenching of electrochemiluminescence upon oligonucleotide hybridization.

Many genomic assays rely on a distance-dependent interaction between luminescent labels, such as luminescence quenching or resonance energy transfer. We studied the interaction between electrochemically excited Ru(bpy)(3) (2+) and Cy5 in a hybridization assay on a chip. The 3' end of an oligonucleotide was labelled with Ru(bpy)(3) (2+) and the 5' end of a complementary strand with Cy5. Upon the hybridization, the electrochemiluminescence (ECL) of Ru(bpy)(3) (2+) was efficiently quenched by Cy5 with a sensitivity down to 30 nmol/L of the Cy5-labelled complementary strand. The quenching efficiency is calculated to be 78%. A similar phenomenon was observed in a comparative study using laser-excitation of Ru(bpy)(3) (2+). The hybridization with the non-labelled complementary or labelled non-complementary strand did not change the intensity of the ECL signal. Resonance energy transfer, electron transfer and static quenching mechanisms are discussed. Our results suggest that static quenching and/or electron transfer are the most likely quenching mechanisms.     

光质对香果树种子萌发及幼苗生长影响的研究

研究了光质对香果树种子萌发及幼苗生长的影响。设置940 nm（远红光）、850 nm（远红光）、730 nm（远红光）、630 nm（红光）、610 nm（橙光）、590 nm（黄光）、525 nm（绿光）、460 nm（蓝光）8个光质处理及自然光对照,研究其种子萌发对光质的响应,设置730、630、610、590、525和460 nm六个光质处理,研究其幼苗生长对光质的响应。结果表明,940及850 nm下无种子萌发,730 nm处理下萌发率仅为1.33%。525 nm下香果树种子萌发率显著高于其他处理及自然光对照,自然光下与630、590 nm下香果树种子最终萌发率无显著差异。可见光中,460及610 nm下香果树种子最终萌发率显著低于其他处理;实验120 d时香果树幼苗干重为590 nm > 630 nm > 610 nm > 730 nm > 525 nm > 460 nm。120 d时,590 nm下香果树幼苗干重显著高于其他光质处理。香果树幼苗的相对质量增长速率,在30~90 d间630 nm显著高于其他处理,在90~120 d间590 nm下显著高于其他处理,在120~150及150~180 d间460 nm下显著高于其他处理。实验30 d时,根重比在0.17~0.25,处理间无显著差异,150 d时,460 nm下根重比显著高于其他处理。实验30 d时,730 nm下茎重比显著高于其他处理。实验30 d时,各处理香果树叶重比在0.53~0.68,处理间无显著差异。     

A fluorometric assay for cyclic guanosine 3',5'-monophosphate incorporating a Sep-Pak cartridge and enzymatic cycling.

We developed a sensitive and nonradioactive fluorometric assay for cyclic guanosine 3',5'-monophosphate (cGMP). Guanine nucleotides except cGMP were enzymatically phosphorylated to GTP. cGMP, absorbed into a Sep-Pak amino propyl cartridge, was eluted separately from GTP. Purified cGMP was enzymatically converted to GTP, which was applied to the GTP-GDP cycle using succinic thiokinase and pyruvate kinase. When pyruvic acid produced by the GTP-GDP cycle was reduced by lactate dehydrogenase, a reduced form of nicotinamide adenine dinucleotide (NADH) was equivalently oxidized to NAD(+). NAD(+) was further converted into fluorescent compound, which was excited at 370 nm and emitted fluorescence at 460 nm, by a strong alkali. When 20 nmol NADH was used for this assay, the calibration curve over 50 to 500 fmol cGMP became sufficiently linear. The detection limit for cGMP was ca. 5 fmol (signal to noise ratio >3). Using this assay, we confirmed that the cGMP content in the left atrial strip of dog was changed from 11.4 +/- 3.8 to 19.3 +/- 2.6 fmol/mg wet wt of tissue (mean +/- SE, n = 6) by electrical driving at 1 Hz. Carbachol (1 microM) further increased the cGMP to 45.6 +/- 9.2 fmol/mg wet wt of tissue. From these results, it is suggested that this novel assay for cGMP is highly sensitive and can be applied to various biological samples.     

Efficient fluorescence energy transfer system between fluorescein isothiocyanate and CdTe quantum dots for the detection of silver ions

We report a fluorescence resonance energy transfer (FRET) system in which the fluorescent donor is fluorescein isothiocyanate (FITC) dye and the fluorescent acceptor is CdTe quantum dot (QDs). Based on FRET quenching theory, we designed a method to detect the concentration of silver ions (Ag⁺). The results revealed a good linear trend over Ag⁺ concentrations in the range 0.01–8.96 nmol/L, a range that was larger than with other methods; the quenching coefficient is 0.442. The FRET mechanism and physical mechanisms responsible for dynamic quenching are also discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

Magic lite design and development

Chemiluminescence immunoassays have now achieved a recognized place in the diagnostic laboratory. The advantages of this non-isotopic technology derive from the use of acridinium esters which can be used to label antigens and antibodies to high specific activities, as well as from optimized immunochemistry. The availability of simple, reliable instrumentation for chemiluminescence measurement together with a range of assay kits offers a logical alternative to traditional radioimmunoassay.     

Two-photon excitation fluorescence resonance energy transfer with small organic molecule as energy donor for bioassay

A fluorescence resonance energy transfer (FRET) model using two-photon excitable small organic molecule DMAHAS as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-Cl. Binding of DABS-Cl labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light, the coexcitation of the energy acceptor with the donor. And such small molecules also show advantages over inorganic up-converting particles that also give anti-Stokes photoluminescence and have been applied as FRET donor recently. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor for FRET-based bioassays.     

How to evaluate the distribution of an "invisible" amphiphile between biological membranes and water

To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzyme's activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range F?rster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.     

Increased Sensitivity of the Microbiological Assay for Biotin by Lactobacillus plantarum

Addition of Tween 80 to biotin assay medium containing acid-hydrolyzed casein as the amino acid source caused marked growth of Lactobacillus plantarum ATCC 8014 in the absence of added biotin. This growth-promoting activity could be eliminated by treating the "vitamin-free" Casamino Acids (Difco) with activated charcoal (Darco G-60) at pH 3.5 for 30 to 60 min. Incorporation of Tween 80 and charcoal-purified Casamino Acids (PCA) into the assay medium (0.8 g and 27 g, respectively, per liter of single strength medium) in place of unpurified Casamino Acids resulted in a medium in which L. plantarum responded to 30 to 50 times less biotin over an extended linear response range (1.3 logs versus 1.0 log) than was required for similar growth in the standard medium. Endogenous growth in the modified medium was absent if the inoculum used was of low density, if it was prepared from biotin-deficient cells, and if the reagents used were free from contaminating traces of biotin. Assays of biological materials for biotin content using the standard medium and the Tween 80-PCA-modified medium resulted in nearly identical values for all samples tested.     

Liposome-based homogeneous luminescence resonance energy transfer

There is an increasing need for developing simple assay formats for biomedical screening purposes. Assays on cell membranes have become important in studies of receptor-ligand interactions and signal pathways. Here luminescence energy transfer was studied on liposomes containing europium ion chelated to 4,4,4-trifluoro-1-(2-naphthalenyl)-1,3-butanedione and trioctylphosphine oxide. Energy transfer efficiency was characterized with biotin-streptavidin interaction, and a model assay concept for a homogeneous time-resolved luminescence resonance energy transfer (LRET) assay was developed. Acceptor-labeled streptavidin was bound to biotinylated lipids on the liposomes, leading to close proximity of the LRET pair. The liposome-based LRET assay was optimized for dye incorporation and concentration, biotinylation degree, liposome size, and kinetics. Sensitivity for a competitive biotin assay was at a picomolar range with a coefficient of variation from 7 to 20%. The developed lipid membrane-based system was feasible in separation free LRET assay concept with high sensitivity, indicating that the assay principle can potentially be used for biologically more relevant target molecules.     

A sensitive and specific radiometric method for the measurement of plasma histamine in normal individuals

A radioenzymatic method for assaying histamine using histamine-N-methyltransferase from guinea pig brain was modified to increase its sensitivity, precision, and specificity. This was achieved by incorporation of a thin-layer chromatography step and use of N_α-methyl histamine as an internal standard in each sample. No prior extraction of the plasma samples is required, permitting the assay of 30 samples in 1 working day. Histamine was detected in the plasma of 17 normal volunteers, at a level of 3.4 ± 0.69 nmol/liter (range 0.82 to 4.7 nmol/liter). In 19 asthmatics, a low-peak expiratory flow rate was found to be associated with a plasma histamine concentration above this “normal” range.     

Purine accumulation in human fat cell suspensions. Evidence that human adipocytes release inosine and hypoxanthine rather than adenosine

Human adipocytes are of limited viability (7 +/- 2% release of lactate dehydrogenase/h) and contain active ectophosphatases which are capable of sequentially degrading ATP to adenosine. At densities of 30,000-40,000 cells/ml, human fat cell suspensions accumulated adenosine, inosine, and hypoxanthine, and their concentrations were 38 +/- 8, 120 +/- 10, and 31 +/- 7 nmol/liter after 3 h of incubation. Dipyridamole (10 mumol/liter), an inhibitor of nucleoside transport, caused a 5-7-fold increase in adenosine accumulation which was reduced by 85% on inhibition of ectophosphatases by beta-glycerophosphate and antibodies against ecto-5'-nucleotidase or alpha, beta-methylene 5'-adenosine diphosphate (10 mumol/liter), respectively, indicating that most of the adenosine is produced in the extracellular compartment. Accordingly, the spontaneous accumulation of adenosine was reduced beyond 5 nmol/liter on inhibition of ectophosphatase activities or removal of extracellular AMP by AMP deaminase (4 units/ml). Added adenosine (30 nmol/liter) disappeared until its concentration approached 5 nmol/liter. Isoproterenol (1 mumol/liter) had no effect on adenosine accumulation regardless whether purine production from extracellular sources was minimized or not. In contrast to adenosine, the concentrations of inosine and hypoxanthine displayed only a modest decrease (30-50%) on inhibition of ectophosphatase activities. In addition, isoproterenol caused a 2-3-fold increase in inosine and hypoxanthine production which was concentration-dependent and could be inhibited by propranolol. It is concluded that the adenosine that accumulates in human adipocyte suspensions is almost exclusively derived from adenine nucleotides which are released by leaking cells. By contrast, inosine and hypoxanthine are produced inside the cells, and the release of these latter purines appears to be linked to ATP turnover via adenylate cyclase.     

Luminescent immobilized enzyme test systems for inorganic pyrophosphate: assays using firefly luciferase and nicotinamide-mononucleotide adenylyl transferase or adenosine-5'-triphosphate sulfurylase.

Inorganic pyrophosphate was measured by luminescence produced by a pyrophosphatase (NAD adenylyl-transferase or ATP sulfurylase) coimmobilized with firefly luciferase on Sepharose beads, with continuous flow of saturating concentrations of substrates (NAD plus luciferin or adenylophosphosulfate plus luciferin, respectively) and intermittent injections of samples containing pyrophosphate. In this scheme, the limiting substrate (pyrophosphate) is regenerated, a situation that is well suited to a bioluminescent assay. The instrumentation allowed for automation with a through-put of approximately one sample every 4 min. With standard solutions or samples that do not contain ATP, the sensitivity of the assay permits detection of less than 1 pmol pyrophosphate in a volume of 20 microliters (50 nmol/liter) with a coefficient of variation approximately equal to 4%. To assay biological samples, it was shown that endogenous ATP can be inactivated by oxidation with sodium periodate. Periodate treatment and quenching engenders dilution that limits the sensitivity to approximately 600 nmol/liter pyrophosphate in the starting material. The assay has been applied to the determination of intracellular pyrophosphate in human lymphocytes and to the measurement of nucleoside-triphosphate pyrophosphohydrolase in human fibroblasts. The variability of the assay was greater with biological samples than with standard samples, with a coefficient of variation of 15.3% in a series of determinations of intracellular pyrophosphate in a series of replicate lymphocyte lysates. Bioluminescent systems of coupled coimmobilized enzymes offer great promise for sensitive, safe, automated assaying of metabolites.