The soluble form of Escherichia coli alkaline phosphatase (ECAP), commercialized calf intestinal alkaline phosphatase (CIAP) and biotinylated CIAP (Biotin-AP) were tested as probes for nonspecific adsorptions of proteins on micro/nano materials represented by magnetic submicron particles (MSP). The nonspecific adsorptions of three candidate probes were evaluated by directly measuring the activities of the adsorbed probes with 4-nitrophenylphosphate. The activities of those three candidate probes exhibited reasonable resistance to detergents, but the presences of >0.1% glycerol in the adsorption systems greatly reduced their nonspecific adsorptions on MSPs. The nonspecific adsorptions of ECAP and Biotin-AP on some MSPs were significant and saturable but became negligible after such MSPs were coated with small zwitterions, bovine serum albumin and/or streptavidin. Only MSPs applicable to chemiluminescence immunoassays showed negligible nonspecific adsorptions of Biotin-AP and ECAP, but all of the tested MSPs exhibited negligible nonspecific adsorptions of CIAP. Therefore, Biotin-AP and ECAP were suitable probes for nonspecific adsorptions of proteins on micro/nano materials.

A labeling reagent against S. japonicum glutathione-S-transferase (sjGST), denoted as Br-I, was designed, prepared and characterized for covalent immobilization of sjGST on magnetic submicron particles (MSP). Br-I had a large hydrophobic moiety for binding to one active site of sjGST, an extended flexible bromoacetylamide moiety for covalent linkage to any of the accessible amino/sulfhydryl groups through nucleophilic substitution. In addition, Br-I had an extended carboxyl group for conjugation with aliphatic primary amines on the MSP, besides a flexible sketch to link those moieties together. Free Br-I was both a substrate/pro-inhibitor and a monovalent irreversible inhibitor of sjGST. There was >75% inactivation of sjGST after half an hour with free Br-I in excess to the sjGST active site, but only sulfhydryl groups far away from the active site were modified when their quantities were comparable. After conjugation to the MSP, Br-I selectively immobilized sjGST in the presence of alkaline phosphatase as a competitor. The treatment of immobilized sjGST with the mixture of free Br-I and GSH reduced unfavorable adsorption of small hydrophobic compounds. Therefore, after conjugation to biomaterials, Br-I showed promise for covalent site-specific immobilization of sjGST-fused targeted proteins.

综述分离非细胞成分对超顺磁亚微米颗粒性能特征的要求、常见制备策略及性能表征方法.

Data in this article are associated with the research article "Ampholytic ion-exchange materials coated with small zwitterion for high-efficacy purification of ionizable soluble biomacromolecules" (Rao et al., 2018) [1]. This article provided data on how to design ampholytic ion-exchange material (AIEM) for the purification of ionizable soluble biomacromolecules for both high activity yields and favorable homogeneity, with two uricases as protein models and a plasmid as DNA model. Data were made publicly available for further analyses.

The present invention discloses a micro/nanomaterial, a product thereof with a surface covalently modified with a hydrophilic material, and a manufacturing method. The surface of the micro/nanomaterial comprises a carboxyl group or potential carboxyl group. The carboxyl group or potential carboxyl group is converted to an active ester. The product with the surface covalently modified with the hydrophilic material is covalently modified by forming an amide bond between the active ester on the surface and a modification agent, wherein the modification agent is a hydrophilic compound and/or a hydrophilic polymer comprising a primary aliphatic amine and/or a secondary aliphatic amine. The manufacturing method comprises: using a carboxyl group and/or potential carboxyl group monomer to generate an adequate amount of carboxyl groups and/or potential carboxyl groups on the surface of a polymeric material, then converting to form an active ester; using an adequately sized modification agent comprising a primary and/or secondary aliphatic amine, potential zwitterion group, and hydrophilic spacer arm to form an amide bond to obtain a covalently modified layer; and regenerating the active ester on the surface of a covalently modified product, then using an amide bond formation process to create a multi-layered covalent modification. High performance coating of a surface of a micro/nanomaterial is achieved by using a medium-sized hydrophilic modification agent. A modified product has significantly reduced non-specific absorption of a biomolecule.

The screening of ligands in mixtures through magnetic recovery of target-ligand complexes needs site-specific immobilization of 6His-tagged targets on magnetic-submicron-particle. Ni2+-NTA-functionalized magnetic-subnnicron-particle is more readily-available than bis-sulfone-functionalized one. The noncovalent conjugates of 6His-tagged targets and Ni2+-NTA-functionalized magnetic-submicron-particle are stable at pH 8.0, but ligand affinities at pH 8.0 may be different from those at pH 7.4. Of full-length human cyclic nucleotide phosphodiesterase 4B2, the inhibition potency of tested neutral compounds was indeed different at pH 7.4 and 8.0. The noncovalent conjugate of 6His-tagged Escherichia coil alkaline phosphatase and Ni2+-NTA-functionalized magnetic-subnnicron-particle released less than 6% of the immobilized enzyme in 3 h at pH 7.4, which enabled the recovery of >90% target-ligand complexes from mixtures of binding reaction. For a 6His-tagged truncated mutant of human cyclic nucleotide phosphodiesterase 4B2 and Ni2+-NTA-functionalized magnetic-subnnicron-particle, the noncovalent conjugate showed similar stability at pH 7.4. After site-specific immobilization on Ni2+-NTA-functionalized and bis-sulfone-functionalized magnetic-submicron-particle, these two 6His-tagged enzymes reserved >90% of their original activities. The use of bis-sulfone-functionalized magnetic-subnnicron-particle caused nonspecific adsorption of those two 6His-tagged enzymes and much longer time for immobilization. Therefore, it was rational to use Ni2+-NTA-MSP for site-specific immobilization of 6His-tagged targets to screen ligands in mixtures.

By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.

A new formulation of the bireagent kit for serum uric acid assay was developed based on the effects of pH on enzyme stability. At 4 °C, half-lives of uricases from Bacillus fastidious and Arthrobacter globiforms were longer than 15 months at pH 9.2, but became shorter at pH below 8.0; half-lives of ascorbate oxidase and peroxidase were comparable at pH 6.5 and 7.0, but became much shorter at pH higher than 7.4. In the new formulation of the bireagent kit, Reagent A contained peroxidase, 4-aminoantipyrine, and ascorbate oxidase in 50 mM phosphate buffer at pH 6.5; Reagent B contained B. fastidious or A. globiforms uricase in 50 mM sodium borate buffer at pH 9.2; Reagents A and B were mixed at 4:1 to produce a final pH from 7.2 to 7.6 for developing a stable color. The new bireagent kit consumed smaller quantities of three enzymes for the same shelf life. With the new bireagent kit, there were linear responses of absorbance at 546 nm to uric acid up to 34 mM in reaction mixtures and a good correlation of uric acid levels in clinical sera with those by a commercial kit, but stronger resistance to ascorbate. Therefore, the new formulation was advantageous.

Maximum activities of 6His-tagged enzyme/mutants from lysates adsorbed on immobilized anti-tag antibody were predicted as specific activities for comparison.

For concomitant enzyme-linked-immunosorbent-assay (ELISA) of two analytes of interest in one well of a microplate via spectrophotometric-dual-enzyme-simultaneous-assay in one solution (SDESA), beta-D-galactosidase on 4-nitro-1-naphthyl-beta-D-galactopyranoside (4NNPG) and alpha-D-glucosidase on 4-nitrophenyl-alpha-D-glucopyranoside (4NPG) were tested as labels with their consistent optimum buffer (sodium phosphate buffer at pH 7.4). Hydrolysis of 4NNPG yielded 4-nitro-1-naphthol exhibiting the longest absorbance peak of 458 nm and the longest isoabsorbance wavelength of 400 nm for absorptivity equal to that of 4NNPG; hydrolysis of 4NPG produced 4-nitrophenol with the longest absorbance peak of about 405 nm. For SDESA, two enzyme reactions were initiated concurrently; absorbance of two chromogenic products was concomitantly measured in one solution via swift alternation between 450 and 405 nm with a Biotek ELX 800 microplate reader, or between 450 nm and a wavelength close to 400 nm with a spectrophotometer; the overlapped absorbance of chromogenic substances was resolved based on the linear additivity of absorbance. For separate assay, just one substrate was used to detect the corresponding label enzyme. During SDESA, initial rates of two glycosidases tolerated negligible interference from the substances involved; the use of the isoabsorbance wavelength of 400 nm to measure absorbance reduced the limit of quantification (LOQ) of alpha-D-glucosidase. Under the same conditions, the LOQ of either of the enzymes via SDESA was consistent with that via separate assay when activities of the other enzyme were varied over the quantifiable range. By ELISA via SDESA with penicillin G and clenbuterol in one sample as two analytes of interest, the content, the coefficient of variation, the limit of detection, LOQ and the quantification range of either of the analytes were consistent with those via separate assay when quantities of the other analyte were varied over the quantifiable range, respectively. Hence, ELISA via SDESA with those two glycosidases as labels has great promise.

目的:合成4-硝基-1-萘磷酸酯（4-nitro-1-naphthyl phosphate,NNPP）为显色底物测定牛小肠黏膜碱性磷酸酶（alka-line phosphatase,ALP）。方法:4-硝基-1-萘酚（4-nitronaphthol,4-NNP）与三氯氧磷反应,经硅胶柱纯化制得NNPP;检测产物吸收跟踪水解过程测定初速度,双倒数法测定米氏常数（Michaelis-Menten constant,Km）。结果:ALP水解NNPP产物最大差吸收峰接近460 nm,等吸收波长接近405 nm。与p-硝基苯酚相比,在pH 6.0~7.0间4-NNP消光系数为其3倍以上,在pH 7.0以上为其2倍以上。ALP对NNPP的Km约12μmol/L而p-硝基苯基磷酸酯的Km约35μmol/L,产物磷酸相对NNPP的竞争性抑制常数接近20μmol/L。ALP催化NNPP水解效率接近水解p-硝基苯基磷酸酯的40%。结论:NNPP可用于测定ALP活性,且有望与作用于p-硝基苯酚类显色底物的其它酶联用实现单通道两种酶同步测定。

PURPOSE:Magnetic submicron particles (MSPs) are pivotal biomaterials for magnetic separations in bioanalyses, but their preparation remains a technical challenge. In this report, a facile one-step coating approach to MSPs suitable for magnetic separations was investigated.METHODS:Polyethylene glycol) (PEG) was derived into PEG-bis-(maleic monoester) and maleic monoester-PEG-succinic monoester as the monomers. Magnetofluids were prepared via chemical co-precipitation and dispersion with the monomers. MSPs were prepared via one-step coating of magnetofluids in a water-in-oil microemulsion system of aerosol-OT and heptane by radical co-polymerization of such monomers.RESULTS:The resulting MSPs contained abundant carboxyl groups, exhibited negligible nonspecific adsorption of common substances and excellent suspension stability, appeared as irregular particles by electronic microscopy, and had submicron sizes of broad distribution by laser scattering. Saturation magnetizations and average particle sizes were affected mainly by the quantities of monomers used for coating magnetofluids, and steric hindrance around carboxyl groups was alleviated by the use of longer monomers of one polymerizable bond for coating. After optimizations, MSPs bearing saturation magnetizations over 46 emu/g, average sizes of 0.32 μm, and titrated carboxyl groups of about 0.21 mmol/g were obtained. After the activation of carboxyl groups on MSPs into N-hydroxysuccinimide ester, biotin was immobilized on MSPs and the resulting biotin-functionalized MSPs isolated the conjugate of streptavidin and alkaline phosphatase at about 2.1 mg/g MSPs; streptavidin was immobilized at about 10 mg/g MSPs and retained 81% ± 18% (n = 5) of the specific activity of the free form.CONCLUSION:The facile approach effectively prepares MSPs for magnetic separations.

Homogenous bioaffinity analysis with tryptophan/tyrosine residues in native proteins as FÖrster-resonance-energy-transfer (FRET) donors is feasible when suitable fluorophors can act as FRET acceptors in ligands (FRET probes) and FRET efficiency in complexes of proteins and FRET probes is high enough. In complexes of proteins and FRET probes, suitable acceptors should have excitation peaks around 335 nm and high rotation freedom, are preferred to have sufficient quantum yields and excitation valleys around 280 nm. In protein binding sites mimicked with mixtures of neutral phosphate buffer and organic solvents, quantum yields of candidate acceptors are altered inconsistently but their excitation peaks show tiny changes. Fluorophores as acceptors in such FRET probes are buried inside glutathione-S-transferase and have low rotation freedom, but are localized on streptavidin surface and display high rotation freedom; FRET efficiency in complexes of streptavidin and its FRET probes is much stronger than that in complexes of glutathione-S-transferase and its FRET probes. Specially, the quantum yield is about 0.70 for free 1-naphthylamine probe in neutral phosphate buffer, about 0.50 for 1-naphthylamine probe bound by streptavidin, and about 0.15 for that bound by glutathione-S-transferase. The quantum yield is about 0.06 for free dansylamide probe, about 0.11 for dansylamide probe bound by streptavidin and about 0.27 for that bound by glutathione-S-transferase. Therefore, 1-naphthylamine and dansylamide are effective acceptors when they localize on surfaces of complexes of proteins and FRET probes.

To characterize streptavidin immobilization on magnetic submicron particles (MSPs), residual streptavidin after magnetic removal of immobilized streptavidin was quantified with N-biotinyl- N′-(1-naphthyl)-ethylenediamine (BNEDA) based on Forster resonance energy transfer. Residual BNEDA after magnetic removal of bound BNEDA was measured by its own fluorescence. Streptavidin was immobilized at about 12 mg per gram of MSPs and easily retained over 50% of its original activity. These assays facilitated optimized streptavidin immobilization on MSPs.

BACKGROUND:For screening a library of enzyme mutants, an efficient and cost-effective method for reliable assay of enzyme activity and a decision method for safe recognition of mutants of higher activity are needed. The comparison of activity concentrations of mutants in lysates of transformed Escherichia coli cells against a threshold is unsafe to recognize mutants of higher activity due to variations of both expression levels of mutant proteins and lysis efficiency of transformed cells. Hence, by a spectrophotometric method after verification to measure uricase activity, specific activity calculated from the level of total proteins in a lysate was tested for recognizing a mutant of higher activity.RESULTS:During uricase reaction, the intermediate 5-hydroxyisourate interferes with the assay of uric acid absorbance, but the measurement of absorbance at 293 nm in alkaline borate buffer was reliable for measuring uricase initial rates within a reasonable range. The level of total proteins in a lysate was determined by the Bradford assay. Polyacrylamide gel electrophoresis analysis supported different relative abundance of uricase mutant proteins in their lysates; activity concentrations of uricase in such lysates positively correlated with levels of total proteins. Receiver-operation-curve analysis of activity concentration or specific activity yielded area-under-the-curve close to 1.00 for recognizing a mutant with > 200% improvement of activity. For a mutant with just about 80% improvement of activity, receiver-operation-curve analysis of specific activity gave area-under-the-curve close to 1.00 while the analysis of activity concentration gave smaller area-under-the-curve. With the mean plus 1.4-fold of the standard deviation of specific activity of a starting material as the threshold, uricase mutants whose activities were improved by more than 80% were recognized with higher sensitivity and specificity.CONCLUSION:Specific activity calculated from the level of total proteins is a favorable index for recognizing an enzyme mutant with small improvement of activity.

Spectrophotometric-dual-enzyme-simultaneous assay in one reaction solution (SDESA) is proposed. SDESA requires the following: (a) Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak (λ(A)) much larger than that of Product B (λ(B)) formed by Enzyme B action on Substrate B; λ(B) is close to the longest isoabsorbance wavelength of Product A and Substrate A (λ(0)); (b) absorbance at λ(A) and λ(0) is quantified via swift alternation of detection wavelengths and corrected on the basis of absorbance additivity; (c) inhibition/activation on either enzyme by any substance is eliminated; (d) Enzyme A is quantified via an integration strategy if levels of Substrate A are lower than the Michaelis constant. Chemometrics of SDESA was tested with γ-glutamyltransferase and lactate-dehydrogenase of complicated kinetics. γ-Glutamyltransferase releases p-nitroaniline from γ-glutamyl-p-nitroaniline with λ(0) at 344 nm and λ(A) close to 405 nm, lactate-dehydrogenase consumes reduced nicotinamide dinucleotide bearing λ(B) at 340 nm. Kinetic analysis of reaction curve yielded lactate-dehydrogenase activity free from inhibition by p-nitroaniline; the linear range of initial rates of γ-glutamyltransferase via the integration strategy, and that of lactate-dehydrogenase after interference elimination, was comparable to those by separate assays, respectively; the quantification limit of either enzyme by SDESA at 25-fold higher activity of the other enzyme remained comparable to that by a separate assay. To test potential application, SDESA of alkaline phosphatase (ALP) and β-D-galactosidase as enzyme-linked-immunoabsorbent assay (ELISA) labels were examined. ALP releases 4-nitro-1-naphthol from 4-nitronaphthyl-1-phosphate with λ(0) at 405 nm and λ(A) at 458 nm, β-D-galactosidase releases 4-nitrophenol from β-D-(4-nitrophenyl)-galactoside with λ(B) at 405 nm. No interference from substrates/products made SDESA of β-galactosidase and ALP simple for ELISA of penicillin G and clenbuterol in one well, and the quantification limit of either hapten was comparable to that via a separate assay. Hence, SDESA is promising.

A resonant-light-scattering (RLS) method was proposed to quantify phosphate for screening inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE). In acidified mixtures of phosphate, papaverine and molybdate, there were aggregates exhibiting micrometre sizes, no absorbance peaks over 360 nm but strong RLS peaks at 392 nm; Mie scattering thus accounted for the RLS signals. When papaverine was added before molybdate to acidified samples of phosphate, RLS signals at 392 nm were stable from 5 to 25 min since the addition of molybdate; after optimization, phosphate from 0.40 to 3.60 μM was quantifiable. This RLS method tolerated 60 mg L(-1) proteins besides common PDE inhibitors and dimethyl sulfoxide in acidified samples of phosphate; the integration of this RLS method with the coupled action of a phosphomonoesterase on PDE product was thus rational to measure PDE activities without the removal of proteins in samples. By quantifying activities of a truncated mutant of human PDE4B2 via this RLS method, Michaelis-Menten constant, inhibition constants of rolipram, papaverine and theophylline varied over three magnitudes and were consistent with those estimated by an improved malachite green assay of phosphate, respectively. Hence, this novel RLS method was promising for screening inhibitors of PDE isozymes.

Objective:To design and screen high-affinity ligands for schistosoma japonicum glutathione S-transferase(GST) as fusion tag.Methods:pGST-MOLUC was induced to express GST in Escherichia coli BL21 under low temperature and GST was purified by glutathione Sepharose 4B affinity chromatography.By the initial velocity method,Km of GST aganist glutathione(GSH) and 1-chloro-2,4-dinitrobenzene(CDNB) was estimated.S-(2,4-dinitrobenzne)-glutathione(GS-DNB),3,5-dimethylbenzoic acid,4-butylbenzoic acid and ethacrynic acid were tested and the inhibition etfect of symmetrical biamide of such aromatic carboxylic acid on GST was examed.Results:Homogenous GST was successfully obtained.GSH and CDNB followed sequential bisubstrate kinetic mechanism with both Km over 0.10 mmol/L.GS-DNB had a competitive Ki of about 5.0 μmol/L against CDNB but a noncompetitive Ki of about 33 μmol/L against GSH.3,5-dimethylbenzoic acid,4-butylbenzoic acid,and ethacrynic acid showed Ki over 0.20 mmol/L,but N,N'-bis-ethacrynyl-1,4-butyldiamine and N,N'-bis-(4-(n-butyl)-benzoyl)-1,4-butyldiamine displayed strong competitive inhibition on this GST against GSH with Ki of 31 nmol/L and(0.61±0.43) μmol/L(n=3),correspondingly.Conclusion:Symmetrical biamide of low-affinity aromatic carboxylic acid linked via short linear chain has promise to be high-affinity inhibitors to single active site of GST.