应用荧光分析法检测酶的研究进展

酶是细胞新陈代谢的基础,酶的检测在生物技术、疾病诊断及药物开发等领域都具有十分重要的意义。在检测酶的诸多方法中,荧光法因其灵敏度高、检测限低等优势发展迅速。以下简述了近年来荧光法在酶检测领域的研究,根据检测方法的不同分为直接荧光检测法和间接荧光检测法,其中直接检测法又根据不同的底物标记及检测机理进行分类。以下介绍了各种方法的应用,并展望了此类方法的前景和发展趋势,为酶工程及生命科学其他领域的相关研究提供信息。     

A pulse-amplitude modulated fluorescence-based method for assessing the effects of photosystem II herbicides on freshwater periphyton

A test was developed that measures in vivo chlorophyll afluorescence variables to assess the apparent sensitivity of freshwaterperiphytic algae to photosystem II inhibitors. Natural periphyticcommunities from rivers were collected on artificial substrata, and theeffects of short-term exposures to two PSII herbicides (atrazine andisoproturon) on the fluorescence parameters were measured with apulse-amplitude modulated fluorometer. The EC50 for each herbicide werecalculated from fluorescence yield indices, and these results were comparedto ¹⁴C-based primary production measurements on the samecommunities. The fluorescence-based method appears to give very reliableestimations of EC50 for each pesticide we tested, ranging from 0.46 to5.18 M and 0.07 to 6.77 M for atrazine and isoproturon,respectively. This method could be used in ecotoxicology monitoringprograms, to detect changes in natural periphyton populations sensitivity,following photosystem II herbicide contamination in rivers or lakes.     

DNAzyme self-assembled gold nanoparticles for determination of metal ions using fluorescence anisotropy assay

Gold nanoparticles can be exploited to facilitate a highly sensitive and selective metal ion detection based on fluorescence anisotropy assay with metal ion-dependent DNA-cleaving DNAzyme. This assay allows rapid and accurate determination of metal ions in aqueous medium at room temperature. The method has been demonstrated for determination of Cu²⁺ and Pb²⁺ ions. The detection sensitivity can be significantly improved to 1 nM by using a “nanoparticle enhancement” approach. Moreover, the assay was also tested in 384-well plates for high-throughput routine determination of toxic metal ions in environmental samples. The method showed distinct advantages over conventional methods in terms of its potential sensitivity, specificity, and ability for rapid response.     

Comparison of the efficacies of loop-mediated isothermal amplification, fluorescence smear microscopy and culture for the diagnosis of tuberculosis

Background

Despite the advent of novel diagnostic techniques, smear microscopy remains as the most practical test available in resource-limited settings for tuberculosis (TB) diagnosis. Due to the low sensitivity of microscopy and the long time required for culture, feasible and accessible rapid diagnostic methods are urgently needed. Loop-mediated Isothermal Amplification (LAMP) is a promising nucleic-acid amplification assay, which could be accessible, cost-effective and more suited for use with unpurified samples.

Methodology/Principal Findings

In the current study, the objective was to assess the efficacy of a LAMP assay for tuberculosis compared with fluorescence smear microscopy as well as Löwenstein-Jensen (LJ) and Mycobacteria Growth Indicator Tube (MGIT) cultures for the diagnosis of pulmonary tuberculosis using sputum samples. Smear microscopy and culture were performed for decontaminated and concentrated sputum from TB suspects and the LAMP was also performed on these specimens. The LAMP and smear microscopy were compared, in series and in parallel, to culture. LAMP and smear microscopy showed sensitivities of 79.5% and 82.1% respectively and specificities of 93.8% and 96.9% respectively, compared to culture. LAMP and smear in series had sensitivity and specificity of 79.5% and 100.0% respectively. LAMP and smear in parallel had sensitivity and specificity of 82.1% and 90.6% respectively.

Conclusions/Significance

The overall efficacies of LAMP and fluorescence smear microscopy in the current study were high and broadly similar. LAMP and smear in series had high specificity (100.0%) and can be used as a rule-in test combination. However, the performance of LAMP in smear negative samples was found to be insufficient.     

Comparison of culture, ELISA and PCR techniques for salmonella detection in faecal samples for cattle, pig and poultry

Background

Performances of different salmonella detection methods were evaluated by applying them to of artificially contaminated faecal specimens from cattle, pigs and poultry. The NMKL71 method, being the standard reference method for detection of salmonella in the official Swedish control program, was compared with the proposed ISO method using MSRV-selective enrichment for culturing, and also with three commercial ELISA- based systems, Bioline Selecta, Bioline Optima and Vidas, a commercial PCR-based method, BAX^® system, and three different strategies using PCR detection using a non-commercial PCR system.

Results

Altogether, 391 samples were tested, and the overall results clearly indicate that, when faeces from all animal species and all serotypes were included, the MSRV performed best, with a calculated accuracy of 99% and a calculated sensitivity of 98%. The second most sensitive and specific method was the BAX^® system, using the modified enrichment protocol as recommended by the manufacturer for faecal samples. However, this protocol includes one additional day of work, as compared with the standard procedure for food sample analysis by the same method. The different strategies for salmonella detection using non-commercial PCR showed a sensitivity and specificity in the same range as the BAX^® method; furthermore, results were obtained more quickly. The various commercial ELISA methods and the NMKL method showed the poorest performance of the methods included in the study, and were closely dependent on the origin of the faeces used and on which salmonella strain was to be detected.

Conclusion

The study showed that the sensitivity of the different methods depended to a great extent on the origin of the faecal matrices and the salmonella strains used to "spike" the samples.     

白斑综合症病毒实时荧光LAMP检测方法的建立及应用

研究利用ESE-Quant tube scanner检测平台, 建立了一套基于环介导等温扩增技术(Loop-Mediated Isothermal Amplification, LAMP)的实时荧光检测方法, 用于白斑综合征病毒(White Spot Syndrome Virus, WSSV)的检测; 并在此基础上, 与巢式PCR、Real-time PCR和其他已发表的4种LAMP方法在检测灵敏度、实际应用方面进行比较. 结果显示, 研究建立的实时荧光LAMP检测方法在63℃恒温反应30min可检测到最低为105倍稀释的基因组DNA模板, 与Real-time PCR检测方法的灵敏度相当, 高于巢式PCR和其他已发表的4种LAMP方法的检测灵敏度; 而且特异性较好, 与传染性皮下及造血组织坏死病毒等5种常见对虾病原DNA均无交叉反应. 通过构建质粒进一步进行灵敏度测试显示, 本研究建立的实时荧光LAMP检测方法最低检测限度为24个拷贝质粒DNA, 检出时间亦为30min. 通过对66份待检样品的检测结果显示, 实时荧光LAMP检测方法的检出阳性率为7.57%, 准确率为100%, 高于其他WSSV的检测方法. 因此, 研究建立的WSSV实时荧光LAMP检测方法, 操作简单, 反应速度快, 特异性好, 灵敏度高, 成本低廉, 可以直观、实时地观察反应的进行情况, 适合对虾养殖现场及诊断实验室的WSSV快速检测.       

2-methylbutanal, a volatile biomarker, for non-invasive surveillance of Proteus

Pathogen detection needs a paradigm shift from time-consuming conventional microbiological and biochemical tests to much simpler identification methods with higher sensitivity and specificity. In this regard, a simple detection method for frequently isolated nosocomial uropathogen, Proteus spp., was developed using the characteristic volatile 2-methylbutanal released in Luria Bertani broth. The instant reaction of the compound with 5-dimethylaminonaphthalene-1-sulfonylhydrazine (DNSH) has been adapted to develop a sensitive fluorescence assay named “ProteAl” (Prote, “Proteus” & Al, “Aldehyde”). The assay was performed by direct addition of the fluorescence reagent to the culture after 7 h of growth. The distinct green fluorescence by Proteus (other organisms show orange fluorescence) served as the simplest and quicker identification test available for Proteus. In the laboratory, it exhibited 100 % specificity and 100 % sensitivity during testing of 95 strains including standard and known clinical isolates representing frequently encountered uropathogens.     

Noninvasive near-infrared imaging of fluorochromes within the brain of live mice: an in vivo phantom study

Near-infrared fluorescence (NIRF) imaging has great potential for studying physiological and pathophysiological processes noninvasively in several locations of the body. In this study, we evaluated the feasibility of NIRF imaging to visualize fluorescent compounds within the brains of live mice commonly used in brain research. To simulate the presence of a molecular NIRF reporter agent at the site of a lesion, we developed a new in vivo phantom model wherein capsules containing different amounts of an NIRF dye (Cy5.5) were stereotactically implanted deep into the left hemispheres of living mice. To precisely locate the implanted capsules, magnetic resonance imaging (MRI) was performed. Fluorescence reflectance imaging (FRI) and transillumination fluorescence imaging (TFI) were conducted to analyze and compare sensitivity and target-to-background ratios of the two methods. The sensitivities of FRI and TFI to background fluorescence from circulating dye was tested by imaging fluorescent capsules in mice intravenously injected with increasing amounts of long-circulating Cy5.5-dextran. The results show that capsules containing dye amounts as low as 10(-12) mol can be detected. TFI yielded significantly higher target-to-background ratios than FRI at 10(-11) mol (p < .05). Comparatively low amounts of fluorescence in the blood vessels can extinguish the signal. We conclude that keeping the signal from circulating NIRF dye low, NIRF imaging offers high sensitivity in detecting fluorochromes noninvasively within brains of mice, especially by using TFI. This encourages the application of NIRF for molecular imaging in the mouse brain using NIRF reporters.     

4-Azido-7-nitrobenzoxadiazole as innovative clickable fluorescence probe for trace and selective quantification of ethinylestradiol in human plasma

Quantification of ethinylestradiol (EE) in biological matrices is challenging as it is a very potent drug with a very low C_max (75 pg.ml⁻¹). Despite the high sensitivity of fluorometric methods, the detection of EE was confined because its structure exhibited very limited fluorescence. Therefore, it must be derivatized first using a fluorogenic agent to produce a more potent fluorescence derivative to achieve the desired ultrasensitive bioanalysis. Here, for the first time, we proposed a promising click fluorescent probe, 4-azido-7-nitrobenzoxadiazole (NBD-AZ) to react with the alkyne group of EE, with the help of copper sulphate and l -ascorbic acid to give a highly fluorescent and stable 1,2,3-triazole derivative. Density functional theory calculation revealed how the triazole formation affects the quantum yield and fluorescence of click reaction product when compared with NBD-AZ. The resulting triazole exhibited a strong signal at a wavelength of 540 nm after excitation at 470 nm. Reaction parameters impacting the intensity of fluorescence were cautiously studied and optimized. The suggested approach has shown outstanding performance, high linearity (25–300 pg.ml⁻¹) and a low detection limit of 7.5 pg.ml⁻¹_. The enhanced sensitivity and selectivity were exploited for analyzing EE in plasma using liquid–liquid extraction for samples cleaning up without interference from any biological components and with a mean % recovery of 100.13 ± 0.39. Accuracy, sensitivity, selectivity, simplicity, and cost–effectiveness make this approach a convincing, promising, and appealing alternative to the reported analytical methods for EE bioanalysis in different matrices.     

Automated detection of elementary calcium release events using the á trous wavelet transform

We developed an algorithm for the automated detection and analysis of elementary Ca2+ release events (ECRE) based on the two-dimensional nondecimated wavelet transform. The transform is computed with the "à trous" algorithm using the cubic B-spline as the basis function and yields a multiresolution analysis of the image. This transform allows for highly efficient noise reduction while preserving signal amplitudes. ECRE detection is performed at the wavelet levels, thus using the whole spectral information contained in the image. The algorithm was tested on synthetic data at different noise levels as well as on experimental data of ECRE. The noise dependence of the statistical properties of the algorithm (detection sensitivity and reliability) was determined from synthetic data and detection parameters were selected to optimize the detection of experimental ECRE. The wavelet-based method shows considerably higher detection sensitivity and less false-positive counts than previously employed methods. It allows a more efficient detection of elementary Ca2+ release events than conventional methods, in particular in the presence of elevated background noise levels. The subsequent analysis of the morphological parameters of ECRE is reliably reproduced by the analysis procedure that is applied to the median filtered raw data. Testing the algorithm more rigorously showed that event parameter histograms (amplitude, rise time, full duration at half-maximum, and full width at half-maximum) were faithfully extracted from synthetic, "in-focus" and "out-of-focus" line scan sparks. Most importantly, ECRE obtained with laser scanning confocal microscopy of chemically skinned mammalian skeletal muscle fibers could be analyzed automatically to reproducibly establish event parameter histograms. In summary, our method provides a new valuable tool for highly reliable automated detection of ECRE in muscle but can also be adapted to other preparations.     

Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines

This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those described in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 x 10(4) to 1 x 10(6) ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio. Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel, sensitive method for determining benzo[a]pyrene-diones using high-performance liquid chromatography with post-column zinc reduction

A novel and sensitive high-performance liquid chromatography (HPLC) method was developed to analyze dione metabolites of benzo[a]pyrene (BaP). Because BaP-diones do not fluoresce, detection of low concentrations is difficult to achieve when analyzing these chemicals with a simple HPLC system. We developed a method to increase the detection sensitivities for BaP-diones using reduction by zinc after the chromatographic separation. A post-column zinc reducer was used to convert BaP-diones, in-line, to their corresponding fluorescent BaP-hydroquinones, which can be measured by fluorescence detection with high sensitivity. With 20-muL injections, the limits of detection for the BaP-diones tested (BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione) were all below 1.0 nM. In addition to the high detection sensitivity, this HPLC method provides a wide linear dynamic range for BaP-dione detection (1.0-220 nM). We also studied the extraction recovery of BaP-diones from recombinant human cytochrome P450 and epoxide hydrolase. To demonstrate the application of this method, the kinetics of BaP-dione formation was studied by incubating BaP with these recombinant enzymes. The present method enhances the detection sensitivity for BaP-diones by more than two orders of magnitude compared with traditional ultraviolet detection. Moreover, the method avoids the time-consuming derivatization or reduction steps required by other methods.     

Comparative evaluation of two enzyme linked immunosorbent assay methods and three Western Blot methods for the diagnosis of culture-confirmed early Lyme borreliosis in Italy

This study investigated the onset and development of the immune response to Borrelia burgdorferi infection in 30 Italian patients with culture-confirmed Lyme Borreliosis in the stage of erythema migrans (EM). All patients received antimicrobial treatment when entering the study and were prospectively evaluated monthly for up to 30 days after enrolment. A total of 60 serially collected serum samples were tested by using two different commercial enzyme-linked immunosorbent assays (ELISAs): Anti-Borrelia plus VlsE ELISA, Euroimmun, and the synthetic peptide-based ELISA, Quick ELISA C6, Immunetics. Sixty-five potentially cross-reacting sera were also tested. Anti-Borrelia plus VlsE ELISA IgG was far more sensitive than Quick ELISA C6 (56.6% and 33.3%, respectively). Moreover, considering that 17 additional sera from the first bleeding group of Lyme disease patients were IgM positive when tested by Anti-Borrelia plus VlsE IgM, the sensitivity of Anti-Borrelia plus VlsE as a whole system rose to 85.0%. Nevertheless, due to the specificity values of Anti-Borrelia plus VlsE ELISA identified in this study (98.5% for IgG and 78.5% for IgM), the need of a confirmatory test for the diagnosis of Lyme disease remains. All the sera were also tested by two different commercial Western Blot (WB) assays: Euroline-WB against Borrelia, Euroimmun, and Qualicode B. burgdorferi WB, Immunetics, in comparison with a multispecies "home made" WB. Performances of the three WB methods for the detection of IgM were very similar. On the contrary, these WBs performed with different values of sensitivity and specificity when IgGs were evaluated. The most sensitive method was the "home-made" WB IgG (71.7%), followed by the Euroline-WB IgG against Borrelia (68.3%). Qualicode B. burgdorferi WB IgG demonstrated to be only 26.6% sensitive. Both "home-made" WB IgG and Qualicode B. burgdorferi WB IgG were 100% specific, whereas Euroline-WB IgG against Borrelia scored 12 cross-reacting samples as borderline, showing a specificity value of 80.0%.     

Modification and optimization of PCR methods for detection of MIE region from human herpesvirus 5

Human herpesvirus 5 (HHV-5, formerly known as CMV) is a beta-herpesvirus widely spread within a population. Thus, HHV-5 infections are a serious matter of concern in a group of immunocompromised patients. The goal of the study was modification and optimization of conventional PCR method developed for the detection of HHV-5 DNA to the real-time variant (RTmPCR) and determination of analytical resolution of the modified methods. Thirty plasma samples were tested for the presence of HHV-5 DNA using the LightCycler system with two different methods--one with SYBR Green I fluorochrome method and second one using TaqMan fluorescent probes and a qualitative in-house gel-stained PCR assay using primers that amplify part of HHV-5 MIE gene. The analytical sensitivity of real-time PCR assay was tested using serial dilutions of HHV-5 DNA in range between 10(0) and 10(-6). For comparison typical end-point detected PCR for cytomegalovirus detection with the same DNA dilutions was made. The sensitivity of novel method was about 100-fold higher than older one. Both LightCycler assays detected HHV-5 DNA in 27 samples, also which were negative by the gel-stained PCR. Analysis of the available clinical and serological data associated with these samples suggested that the real-time results in all of these cases were true positive. The conclusion is that real-time PCR methods are more sensitive than the conventional PCR used in this study. The additional sensitivity was valuable for detection of patients with low-copy viremia. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler instrument are favorable for the use of this system in the detection of HHV-5 DNA in clinical specimens.     

Flow cytometric analysis of experimental parameters for the immunofluorescent labeling of BrdUrd in various tumour cell lines

Summary This report describes the results of the comparison of three different methods and three monoclonal antibodies to stain cells in suspension for incorporated bromodeoxyuridine and total DNA content. The procedures were tested in three different experimental tumour cell lines. The sensitivity of the different procedures was expressed as the ratio of the anti-BrdUrd fluorescence intensities of the S and G1 phase cells (FS/FG1 ratio). There were remarkable differences in sensitivity between the different procedures. With the heat denaturation the most favourable FS/FG1 ratio's were obtained but substantial cell loss occurred during this procedure which is a disadvantage for clinical application. With the pepsin digestion + acid denaturation procedure cell loss was negligible. The standard acid denaturation procedure was inferior to the other two methods. Using the pepsin digestion + acid denaturation procedure we examined the variations in sensitivity for the different monoclonal antibodies and cell lines and the influence of BrdUrd concentration, labelingtime and cell concentration. The binding characteristics for the various antibodies differed considerably in our hands. Only with the IU4 antibody we obtained FS/FG1 ratio's comparable with those desenbed in the literature. No difference was observed between the cell lines. Variation in cell concentration between 1 × 10⁴ to 1 × 10⁶ ml nor BrdUrd concentration appeared to influence the sensitivity of the procedure. A labelingtime of 1 h or even 30 min seems to be more than sufficient for an optimal FS/FG1 ratio.Our results indicate that using the appropriate antibody and immunofluorescence BrdUrd can be detected by flow cytometry, after incorporation into the DNA of tumour cells under a wide range of culture conditions.For clinical application, the pepsin digestion + acid dena uration method in combination with IU4 antibody seems to be the procedure of choice due to its good reproducibility, sensitivity and its low cell loss.     

解离增强镧系元素荧光免疫分析灵敏度的改进

为提高解离增强镧系荧光免疫分析(DELFIA)的灵敏度或信／噪比,进行了一些重要的方法学研究．观察到了对于不同的铕量,荧光响应和信／噪比都随着增强液体积而明显地变化．对于确定的铕量,存在一个最佳体积,且铕量越小,其最佳体积也越小．实验中选择最佳体积是重要的．研究了增强液制备技术,发展了微滴定板条有效的清洗和干燥方法,使本底荧光明显降低．     

Fluorescence fading and stabilization in cytofluorometry

The factors affecting fluorescence fading in cytofluorometry were investigated using different kinds of nuclear staining, mounting media, and procedure of specimen preparation. Acceleration of fluorescence fading was observed in smear specimens treated with RNase, trypsin, or hypotonic solution before pararosaniline Feulgen nuclear staining. Similar effect was found for other DNA-stainings such as "33258 Hoechst" and Feulgen reactions with different Schiff-type dyes, such as acriflavine-SO2 and cresyl-violet-SO2, when chemically pure DNA was used. Fluorescence decay was rapid for all fluorochromes examined, when glycerin or buffer solution was used as mounting medium. Marked stabilization of fluorescence emission was induced in specimen mounted in non-fluorescent resin, Entellan (Merck), after post-staining fixation with absolute methanol for all tested fluorochromes. The same treatment induced almost complete fluorescence stabilization of fluorescein isothiocyanate (FITC); no detectable fluorescence fading was observed in a specimen stained with indirect immunofluorescence reaction using anti-UV-DNA antibody, during storage for 2 years at room temperature without special protection against light. These observations suggest that factors which bring about conformational stability of macromolecule-dye complexes generally induce fluorescence stabilization.     

Highly sensitive and selective derivatization-LC method for biomolecules based on fluorescence interactions and fluorous separations

A fluorescence derivatization LC method is a powerful tool for the analysis with high sensitivity and selectivity of biological compounds. In this review, we introduce new types of fluorescence derivatization LC analysis methods. These are (1) detection-selective derivatization methods based on fluorescence interactions generated from fluorescently labeled analytes: excimer fluorescence derivatization and fluorescence resonance energy transfer (FRET) derivatization; (2) separation-selective derivatization methods using the fluorous separation technique: fluorous derivatization, F-trap fluorescence derivatization, and fluorous scavenging derivatization (FSD).     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.