口香糖中人参总皂甙样品前处理方法比较研究

用乙醚分散口香糖胶基,温水超声提取,水饱和正丁醇萃取,可见分光光度法对样品进行定量测定。与人参皂甙测定的行业标准方法相比,本方法样品前处理方法更有效,样品测定结果线性好(r=0.9993),平均回收率为99.2%,精密度实验RSD为1.7%(n=6)。本方法可以作为口香糖中人参总皂甙分析测定的简便、快速方法。     

基于脉动变光程的分光光度法自标定检测磷酸盐

分光光度法应用于磷酸盐长期监测存在络合物对透光窗口附着污染的干扰问题.本研究采用新的脉动变光程的分光光度检测原理,提出自标定检测方法,开发了脉动可变光程的机械装置.利用此设备实现了水体磷酸盐检测的长期原位自标定.利用本方法与传统分光光度计进行长期监测对比实验,结果表明:本方法能够有效去除镜头附着污染等干扰因素影响,提高了检测结果的准确性与可靠性.     

茚三酮显色法测定壳聚糖含量

本研究根据壳聚糖与茚三酮作用显色的特性,建立可见光分光光度法测定壳聚糖含量的方法,并对该测定方法的适宜条件及工作曲线进行确定。实验结果表明,壳聚糖与茚三酮在p H5.5的HAc-Na Ac缓冲体系中显色稳定,在0.004~0.040 g·L-1浓度范围内,其复合物在570nm处吸光度与壳聚糖浓度线性关系良好(r2=0.9981)。进一步对该方法的稳定性、重现性和回收率进行考察,表明该方法操作简单,结果可靠,可满足样品中微量壳聚糖含量的常规检测需要。     

三重-比导数分光光度法及其应用研究

提出了可以应用于三元混合物同时测定的三重 比导数分光光度法,阐述了三重 比导数分光光度法的基本原理,研究了三重 比导数分光光度法在三元复方制剂增效联磺片中磺胺甲口恶唑(SMZ)、磺胺嘧啶(SD)、甲氧苄啶(TMP)的同时测定。试验结果表明,增效联磺片中SMZ、SD、TMP的回收率分别为102 1%(RSD=2.7%,n=20)、100.5%(RSD=1.8%,n=20)、101.2%(RSD=4 3%,n=20)。     

改进分光光度法快速测定锡精矿中的砷

建立了改进的分光光度法快速测定锡精矿中砷的方法,样品经过氯酸钾-硝酸-盐酸低温溶解、硫酸发烟处理,然后用盐酸溶解后澄清,取清液用分光光度法进行砷的测定。方法简便、实用、快速。经国家标准物质分析验证,测定值与标准值相符,相对标准偏差(RSD,n=12)为1.1%~1.4%;适用于锡精矿中0.01%以上砷的测定。     

抗坏血酸有机锗倍半氧化物油水分配系数的测定

抗坏血酸有机锗倍半氧化物是一种抗肿瘤药物。通过建立了紫外分光光度法测定抗坏血酸有机锗倍半氧化物含量的方法,用摇瓶法测定其油水分配系数。建立标准曲线方程A=5.312 9C+0.043 0,检测波长217nm,回收率为98.28%,油水分配系数P=0.37。应用摇瓶-紫外分光光度法,能够准确测定抗坏血酸有机锗倍半氧化物的油水分配系数,为脂质体处方设计提供参考。     

ICP-AES法检测工业用精对苯二甲酸中金属元素

研究用ICP-AES法同时测定工业用精对苯二甲酸中铬、钴、铁、锰、钼、镍、钛等金属元素.通过实验确定了入射功率、冷却气流量、辅助气流量、雾化压力等实验条件.该方法的检出限为0.0013～0.0148 mg/L,测定结果的相对标准偏差为0.16%～0.30%(n=11),加标回收率为90.6%～96.2%.该方法对试样的测定结果与原子吸收分光光度法及二安替比林甲烷分光光度法的检测结果基本一致.     

荧光光度法测定瑞舒伐他汀钙含量的研究

优化荧光分光光度法测定瑞舒伐他汀钙含量的方法,建立方法学,并用紫外-分光光度法验证结果的可靠性。在激发波长250nm,发射波长516nm的条件下采用荧光分光光度法测定瑞舒伐他汀钙片中的瑞舒伐他汀钙的含量。以荧光强度为指标,考察pH值、甲醇浓度和时间等3个因素,选定最佳条件,结果的准确性和可靠性用紫外-分光光度法进行验证。荧光强度与瑞舒伐他汀钙浓度在3.17～39.72mg·L~(-1)范围内具有良好的线性关系,回归方程F=13.96C+145.71(r=0.999 8),检出限为0.38mg·L~(-1),平均回收率为98.4%,RSD 1.05%。最佳实验条件为:pH值6.7,时间10 min,甲醇浓度为95%。对正交试验数据进行统计分析,具有统计学意义(P0.05)。结果显示用荧光分光光度法测定瑞舒伐他汀钙含量特异性好,灵敏度高,检测限低。采用紫外-分光光度法进行验证,结果一致。     

微波消解/干灰化-分光光度法测定面制食品中的铝

采用微波消解法和干灰化法对面制品进行前处理,用分光光度法测定面制品中的铝含量,建立了微波消解/干灰化-分光光度法测定铝的方法.实验结果表明,本方法前处理简单,检测结果准确度高,精密度好,在0～10μg/25 mL线性范围内:相关系数r=0.999 5,方法检出限为0.2 μg/25 mL,样品的加标回收率在92.0 %...     

对苯二胺-铁(Ⅲ)体系荧光分光光度法检测水样中痕量硫化氢

建立了对苯二胺-Fe(Ⅲ)体系荧光分光光度法快速有效测定水样中痕量H_2S的新方法。在酸性体系中,对苯二胺与FeCl_3及H_2S反应产生有荧光的劳氏紫,用荧光分光光度法进行测定。其激发波长和发射波长分别为590 nm和620 nm,1.3~65μmol/L,相关系数r=0.9982,检出限为0.122μmol/L,回收率为97.7%~113.2%。方法适用于水样中痕量H_2S含量的测定。     

Dipicolinic acid (DPA) assay revisited and appraised for spore detection

Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 microM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10(4) Bacillus subtilis spores ml-1 was reached, representing a substantial improvement over previous rapid tests.     

EFFECT OF DIPICOLINIC ACID ON THE ULTRAVIOLET RADIATION RESISTANCE OF BACILLUS CEREUS SPORES

Abstract— The ultraviolet radiation (UV) resistance of B. cereus spores was shown to depend on their content of dipicolinic acid (DPA). Wild-type spores with decreasing amounts of DPA exhibited increased UV resistance. Similarly, spores devoid of DPA (DPA-minus), produced by a mutant strain of B. cereus unable to synthesize DPA, were more resistant to UV than mutant spores (DPA-plus) produced in the presence of exogenously supplied DPA. Resistance of both the wild type and mutant strains to ionizing radiation, however, was unaffected by DPA content. Comparison of the resistance of DPA-minus and DPA-plus mutant spores to UV of various wavelengths showed that the greater sensitivity of the latter DPA-plus spores appeared at wavelengths corresponding to the region of the first molecular absorption band of the calcium chelate of DPA. In the wild type and mutant, thymine photoproducts were produced at a greater rate and to a greater extent in spores with high levels of DPA than in spores with low DPA.
The data indicate that DPA transfers energy to DN A in vivo , which leads to the conclusion that DPA occurs in the spore protoplast.     

Biosynthesis of Silver Nanoparticles by Bacillus stratosphericus Spores and the Role of Dipicolinic Acid in This Process

Seeking for simple, rapid, and environmental-friendly routes to produce metal nanoparticles is quite attractive for various biotechnological applications. Biological synthesis method of silver nanoparticles has been found very promising due to their non-toxicity and simplicity. Here, the spores of Bacillus stratosphericus isolated from soil enriched with 30 % H₂O₂ were used for the production of silver nanoparticles. Furthermore, the possible mechanism of silver nanoparticle synthesis by the spores was elucidated for the first time. In this regard, dipicolinic acid (DPA) was shown to play a critical role as a nanoparticle-producing agent. UV–Vis absorption spectroscopy, X-ray diffraction technique, energy-dispersive spectroscopy, and transmission electron microscopy were used to characterize the nanoparticles. Unlike vegetative cells of B. stratosphericus, the spores and the purified DPA were capable of producing nanoparticles from silver nitrate (AgNO₃). These biogenic nanoparticles, which were highly toxic against different pathogenic bacteria, showed mixed structures including spherical, triangular, cubic, and hexagonal with the approximate size between 2 and 20 nm in diameter. Our results illustrated the role of dipicolinic acid as a main factor for the synthesis of nanoparticles by the bacterial spores.     

Raman spectra of dipicolinic acid in crystalline and liquid environments

Raman spectra of dipicolinic acid (DPA) are important for detection of bacterial spores, since DPA and its salts present one of their major components. The implementation of a deeply cooled CCD camera in combination with pulsed excitation at 532 nm allowed measuring well-resolved Raman spectra of the DPA in different forms. Powder preparations, crystals grown from saturated solutions and aqueous solutions of the DPA were studied. The spectral features in different environments and comparison with the spectra obtained by other methods are discussed.     

Hydroxyapatite nanoparticle based fluorometric turn-on determination of dipicolinic acid,a biomarker of bacterial spores

Hydroxyapatite nanoparticles (HAP-NPs) were rendered fluorescence by doping with Eu(III) ion. The resulting fluorescent NPs are shown to be viable probes for sensitive and selective determination of dipicolinic acid (DPA), a major constituent of bacterial spores as used in bioterrorism. It is found that the addition of DPA to solutions of such HAP-NPs result in an enhancement of fluorescence due to the coordination of DPA with the Eu(III) dopant. The assay allows DPA to be detected in the 0.1 to 40 μM concentration range and with a 77 nM detection limit. The assay was applied to the detection of spores of Bacillus subtilis. The attractive properties of the probe make it a promising candidate for used in rapid detection of pathogenic bacterial spores.

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Graphical abstract Fluorescent hydroxyapatite nanoparticles (HAP-NPs) are shown to be a viable probe for detection of dipicolinic acid, a major constituent of bacterial spores. The red asterisks represent the fluorescence intensity of the HAP-NPs.

     

Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis

A new capillary zone electrophoresis (CZE) method for the analysis of dipicolinic acid, a specific component found in spores but not in vegetative cells, was used to determine spore concentration in Bacillus thuringiensis according to the relationship between the spore concentration and the content of dipicolinate. The quantitative relationship was established by using purified spores. Electrolyte conditions that affected the separation efficiency of dipicolinate and the reproducibility were investigated. With 10 mM phosphate, 10 mM ethylenediaminetetraacetic acid and 0.25 mM tetradecyltrimethylammonium bromide at pH 6.2 as the carrier electrolyte, dipicolinate can be determined within 8 min at an applied voltage of -25 kV (anode at detector) and a capillary temperature of 25 degrees C. The method has a high separation efficiency with which the number of theoretical plates is above 300,000 plates m(-1). The relative standard deviations for migration time and peak area are less than 0.5% and 2.0%, respectively. The detection limit for dipicolinate was 10 ng ml(-1), which corresponds to 7.2 x 10(5) spores ml(-1). The method was used to determine spores in fermentation broths, and the results obtained agreed well with the values obtained by plate counting.     

Nanoparticle-based substrates for surface-enhanced Raman scattering detection of bacterial spores

The development of ultrasensitive and rapid methods for the detection of bacterial spores is important for medical diagnostics of infectious diseases. While Surface-Enhanced Raman Spectroscopic (SERS) techniques have been increasingly demonstrated for achieving this goal, a key challenge is the development of sensitive and stable SERS substrates or probes. This Minireview highlights recent progress in exploring metal nanoparticle-based substrates, especially gold nanoparticle-based substrates, for the detection of biomarkers released from bacterial spores. One recent example involves assemblies of gold nanoparticles on a gold substrate for the highly sensitive detection of dipicolinic acid (DPA), a biomarker for bacterial spores such as Bacillus anthracis. This type of substrate exploits a strong SERS effect produced by the particle-particle and particle-substrate plasmonic coupling. It is capable of accurate speciation of the biomarker but also selective detection under various reactive or non-reactive conditions. In the case of detecting Bacillus subtilis spores, the limit of detection is quite comparable (0.1 ppb for DPA, and 1.5 × 10(9) spores per L (or 2.5 × 10(-14) M)) with those obtained using silver nanoparticle-based substrates. Implications of the recent findings for improving the gold nanoparticle-based SERS substrates with ultrahigh sensitivity for the detection of bacterial spores are also discussed.     

Direct mass spectrometric analysis of Bacillus spores

Spores from the Bacillus species, B. cereus, B. anthracis, B. thuringensis, B. lichenformis, B. globigi, and B. subtilis, were examined by direct probe mass spectrometry using electron ionization (EI) and positive and negative chemical ionization (CI). Molecular ions from free fatty acids and nucleic acids were observed in the 70eV spectra as were fragments from glycerides. Spectra obtained with isobutane positive chemical ionization (CI(+)) were dominated by ions associated with pyranose compounds such as N-acetylglucosamine (NAG). Unlike the positive ion spectra, the negative ion spectra of the spores were very simple and contained few peaks. The M(-.) ion from dipicolinic acid (DPA) was the base peak in the negative ion spectra of all spore species except those from B. lichenformis. The negative ion of DPA produced such a strong signal that 10(8) colony forming units (CFUs) of B. cereus spores could be detected directly in 0.5 g of ground rice. Principal component analysis (PCA) of the spectra revealed that only CI(+) spectra contained differences that could be used to identify the spectra by species. Differentiation of the CI(+) spectra by PCA was attributed to variances in the peaks associated with the bacterial polymer poly(3-hydroxybutyrate) (PHB) and NAG. Similar differences in PHB and NAG peaks were detected in the CI(+) spectra of a suite of vegetative Bacillus stains grown with various media.     

Electrical detection of germination of viable model Bacillus anthracis spores in microfluidic biochips

In this paper, we present a new impedance-based method to detect viable spores by electrically detecting their germination in real time within microfluidic biochips. We used Bacillus anthracis Sterne spores as the model organism. During germination, the spores release polar and ionic chemicals, such as dipicolinic acid (DPA), calcium ions, phosphate ions, and amino acids, which correspondingly increase the electrical conductivity of the medium in which the spores are suspended. We first present macro-scale measurements demonstrating that the germination of spores can be electrically detected at a concentration of 10(9) spores ml(-1) in sample volumes of 5 ml, by monitoring changes in the solution conductivity. Germination was induced by introducing an optimized germinant solution consisting of 10 mM L-alanine and 2 mM inosine. We then translated these results to a micro-fluidic biochip, which was a three-layer device: one layer of polydimethylsiloxane (PDMS) with valves, a second layer of PDMS with micro-fluidic channels and chambers, and the third layer with metal electrodes deposited on a pyrex substrate. Dielectrophoresis (DEP) was used to trap and concentrate the spores at the electrodes with greater than 90% efficiency, at a solution flow rate of 0.2 microl min(-1) with concentration factors between 107-109 spores ml(-1), from sample volumes of 1-5 microl. The spores were captured by DEP in deionized water within 1 min (total volume used ranged from 0.02 microl to 0.2 microl), and then germinant solution was introduced to the flow stream. The detection sensitivity was demonstrated to be as low as about a hundred spores in 0.1 nl, which is equivalent to a macroscale detection limit of approximately 10(9) spores ml(-1). We believe that this is the first demonstration of this application in microfluidic and BioMEMS devices.     

Analysis of the Raman spectra of Ca(2+)-dipicolinic acid alone and in the bacterial spore core in both aqueous and dehydrated environments

The core of dormant bacterial spores suspended in water contains a large depot of dipicolinic acid (DPA) chelated with divalent cations, predominantly Ca(2+) (CaDPA), and surrounded by water molecules. Since the intensities of the vibration bands of CaDPA molecules depend significantly on the water content in the CaDPA's environment, the Raman spectra of CaDPA in spores may allow the determination of the spore core's hydration state. We have measured Raman spectra of single spores of three Bacillus species in different hydration states including the spores suspended in water, air-dried and vacuum-dried. As a comparison, we also measured the Raman spectra of CaDPA and DPA in different forms including in aqueous solution, and as amorphous powder and crystalline form. We also monitored changes in Raman spectra of an individual spore during dehydration under vacuum. The results indicated that (1) the state of CaDPA in the core of a spore suspended in water is close to an amorphous solid or a glassy state, but still mixed with water molecules; (2) the ratio of intensities of Raman bands at 1575 and 1017 cm(-1) (I(1575)/I(1017)) is sensitive to the water content in the CaDPA's environment; (3) variations in I(1575)/I(1017) are small (～4%) in a population of dormant Bacillus spores suspended in water; and (4) the I(1575)/I(1017) ratio increases significantly during dehydration under vacuum. Consequently, measurement of the I(1575)/I(1017) ratio of CaDPA in spores may allow a qualitative estimation of the degree of hydration of the bacterial spore's core.