Detection of Melamine in Feed Using Liquid-Liquid Extraction Treatment Combined with Surface-Enhanced Raman Scattering Spectroscopy

A rapid, selective, and sensitive method to determine the melamine content in animal feeds was developed using surface-enhanced Raman scattering spectroscopy on aggregated 55 nm Au nanoparticles with liquid–liquid extraction sample preparation. Butyl alcohol was used as the initial extraction solvent, and liquid–liquid extraction was performed twice using HCl (pH 3–4) and 6∶1 (v/v) n-butyl alcohol/ethyl acetate. The intensity of the matrix-based peak at 731 cm⁻¹ was set at 100 as a basis for the feeds, and the peak at 707 cm⁻¹ was the characteristic peak of melamine used in the calculations. Sufficient linearity was obtained in the range 2–10 µg·g⁻¹ (R² = 0.991). Limits of detection and quantification in the feeds were 0.5 and 2 µg·g⁻¹, respectively. The recovery rates were 82.5–90.2% with coefficients of variation below 4.02%. This new protocol could be easily developed for the routine monitoring of on-site feed quality and market surveillance.     

Highly Sensitive Plasmonic Sensor Based on Fano Resonance from Silver Nanoparticle Heterodimer Array on a Thin Silver Film

We investigate the optical spectrum of a multilayer metallic slab using multiple-scattering formalism. A thin silver film is attached to a periodic array of heterodimers consisting of two vertically spaced silver nanoparticles of different radii. Depending on the radius of nanoparticles, heterodimer array presents a simple nanoscale geometry which gives rise to remarkable plasmonic properties of multipolar resonances. Due to the coherent interference of the localized nanoparticle plasmons (discrete mode) and surface plasmon polaritons of metallic film (continuous mode), the reflection spectrum represents a sharp asymmetric Fano resonance dip, which is strongly sensitive to the refractive index of the surrounding embedded dielectric host. The physical features contribute to a highly efficient plasmonic sensor for refractive index sensing with sensitivity of ～1.5?×?10^?3 RIU/nm.     

顶空及SERS结合快速检测葱属植物-大葱挥发物

建立了常温常压下快速检测新鲜葱属植物-大葱主要挥发性气体的方法。采用顶空瓶在常温常压下收集大葱的挥发物,将挥发物用注射器注入纳米银胶中,进行SERS测量。结果表明大葱的挥发物SERS光谱重现性非常好;将大葱挥发性物的SERS谱与1-丙硫醇(1-Propanethiol)和烯丙基甲基硫醚(allyl methyl sulfide)混合气体的SERS谱相比,具有较好的相似性,说明大葱的挥发物主要由1-丙硫醇和烯丙基甲基硫醚气体组成。利用Gaussian 03软件获得1-丙硫醇-银(1-Propanethiol-Ag)的Raman光谱,计算结果与1-丙硫醇的SERS实验结果对应较好,说明1-丙硫醇在纳米银基底上的增强为化学增强。顶空与SERS结合可直接用于对葱属植物挥发性物的研究。     

A Simple and Sensitive Resonance Scattering Spectral Assay for Detection of Melamine Using Aptamer-Modified Nanosilver Probe

Nanosilver of 10-nm size was prepared by the NaBH₄–sodium citrate procedure, and it was modified by a single-strand DNA (ssDNA) aptamer to fabricate an AgssDNA probe for melamine. The probe was stabile at pH 7.0 Na₂HPO₄–NaH₂PO₄ buffer solutions and in the presence of 25.0 mmol/L NaCl. Upon the addition of melamine, it interacted with the probe to aggregate big clusters, which led to the resonance scattering (RS) intensity at 470 nm increasing greatly. Under the selected conditions, the increased RS intensity (ΔI _470 nm) is linear to melamine concentration in the range of 6.31–378.4 μg/L, with a regression equation of DI_{470 nm} = 1.124c + 10.8 \Delta {I_{{47}0{\rm{ nm}}}} = {1}.{124}c + { 10}.{8} and a detection limit of 3.1 μg/L. The aptamer-modified nanosilver RS assay has been applied for the determination of melamine in milk, with satisfactory results.     

Highly Sensitive Resonance Scattering Detection of DNA Hybridization Using Aptamer-Modified Gold Nanopaticle as Catalyst

Gold nanoparticle particles in size of 10 nm were used to label the thiol-modified single-stranded DNA aptamer (SH-ssDNA) to obtain an aptamer-modified gold nanoparticle probe (AussDNA) for target DNA (tDNA). In pH 7.4 NaH₂PO₄–Na₂HPO₄ buffer solution, the hybridization reaction between AussDNA and tDNA took place to form larger aptamer-modified gold nanoparticle cluster complex. The excess aptamer-modified gold nanoparticle probe in the supernatant solutions was obtained by centrifuging and can be used as nanocatalyst for the 0.276 mmol/L CuSO₄-65.4 mmol/L potassium-sodium tartrate-0.37 mmol/L glucose system at 70 °C. The cubic Cu₂O particles generated by the nanocatalytic reducing exhibit a strong resonance scattering (RS) peak at 620 nm. In the selected conditions, the RS intensity at 620 nm decreased with addition of tDNA, and the decreased intensity ΔI _{620 nm} is proportional to tDNA concentration (C _tDNA) from 0.12 to 72 pM, with regress equation of ΔI _620 nm = 1.29C _tDNA + 4.05, correlation coefficient of 0.9917, and detection limit of 0.084 pM tDNA.     

Highly Sensitive Dual-core Photonic Crystal Fiber Based on a Surface Plasmon Resonance Sensor with Gold Film

Plasmonics - A highly sensitive surface plasmon resonance (SPR) sensor comprising a dual-core photonic crystal fiber (PCF) is designed to detect minute changes in analyte refractive indices (RIs)...     

Analysis and Simulation of a Novel Localized Surface Plasmonic Highly Sensitive Refractive Index Sensor

Dividing a metal nanoparticle into smaller components and the occurrence of the plasmonic phenomenon in the gap between these components can improve the sensitivity of the detector to variation of the refraction coefficient of liquid. In this paper, in a constant volume of metal, a golden disk is divided into two rings and one smaller disk. With a proper arrangement of these components, the surface plasmon resonance phenomenon takes place at the wavelength of 945.7 nm. The occurrence of this phenomenon increases the field in the distance between nanoparticles surrounded by liquid. The sensitivity of the detector that designed using nanodisks is 300 nm/RIU while it increases to 500 nm/RIU for the new structure. The increase of LSPR displacement, for a variation of 0.01 in the liquid refraction coefficient, from 3 nm for a disk to 5 nm for a proposed structure verifies a 67% improvement in the sensitivity of the sensor.

     

Design of a Wide Ranging Highly Sensitive Pressure Sensor Based on Two-Dimensional Photonic Crystals

Plasmonics - A highly sensitive pressure sensor operating over a wide pressure range based on two-dimensional photonic crystals having a Mach-Zehnder interferometer structure has been developed....     

Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

Sensitive Detection of Phosphorus Deficiency in Plants Using Chlorophyll a Fluorescence

Phosphorus (P) is a finite natural resource and an essential plant macronutrient with major impact on crop productivity and global food security. Here, we demonstrate that time-resolved chlorophyll a fluorescence is a unique tool to monitor bioactive P in plants and can be used to detect latent P deficiency. When plants suffer from P deficiency, the shape of the time-dependent fluorescence transients is altered distinctively, as the so-called I step gradually straightens and eventually disappears. This effect is shown to be fully reversible, as P resupply leads to a rapid restoration of the I step. The fading I step suggests that the electron transport at photosystem I (PSI) is affected in P-deficient plants. This is corroborated by the observation that differences at the I step in chlorophyll a fluorescence transients from healthy and P-deficient plants can be completely eliminated through prior reduction of PSI by far-red illumination. Moreover, it is observed that the barley (Hordeum vulgare) mutant Viridis-zb⁶³, which is devoid of PSI activity, similarly does not display the I step. Among the essential plant nutrients, the effect of P deficiency is shown to be specific and sufficiently sensitive to enable rapid in situ determination of latent P deficiency across different plant species, thereby providing a unique tool for timely remediation of P deficiency in agriculture.The world population is estimated to exceed 9 billion people by 2050. This means that agriculture on a global scale has to increase food production by 70% to 100%, and, at the same time, handle the consequences of global climate changes and reduce its environmental footprint (Food and Agriculture Organization of the United Nations, 2009; Godfray et al., 2010; Foley et al., 2011). A major challenge related to this is the supply and use of phosphorus (P) to support future plant production (Cordell et al., 2009; Gilbert, 2009; MacDonald et al., 2011).P is an essential plant nutrient, which means that plants require P in adequate amounts to fulfill a complete lifecycle. It has been estimated that 30% of the world’s agricultural soils are P deficient and need fertilizer addition to ensure yield and quality (MacDonald et al., 2011). However, phosphate rock, the main source of P fertilizers, is a finite natural resource, and the known rock phosphate reserves are estimated to last as little as 50 years in the gloomiest forecasts (Gilbert, 2009; Edixhoven et al., 2013). This makes P a potential strategic natural resource similar to oil, as very few countries control the vast majority of the known reserves (Gilbert, 2009; Elser and Bennett, 2011; Edixhoven et al., 2013). Presently, an immense overuse of P is found in some parts of the world, causing eutrophication of lakes and seas, while P depletion results in severe yield limitations elsewhere (MacDonald et al., 2011; Obersteiner et al., 2013). An essential aspect of solving both of these problems is to increase P use efficiency in agriculture, thus reducing the negative environmental impact of agriculture and helping to ensure a sustainable use of P resources while increasing the worldwide food production (Schröder et al., 2011; Veneklaas et al., 2012).Here, we present a unique analytical principle based on chlorophyll a fluorescence that allows rapid, nondestructive, onsite assessment of plant P status by recording the so-called OJIP transient of a dark-adapted leaf.When a chlorophyll molecule absorbs light, one of three events will occur: The light may be used to drive photosynthesis, it can be dissipated as heat, or it can be reemitted as fluorescence. Less than 10% of light absorbed by the plant causes emission of chlorophyll a fluorescence (Govindjee, 2004; Stirbet and Govindjee, 2011). When a dark-adapted leaf is exposed to saturating actinic light, the resulting time-dependent fluorescence forms a so-called Kautsky curve (Kautsky and Hirsch, 1931; McAlister and Myers, 1940). Within 300 ms, the fluorescence increases from a minimum level (F₀) to the maximum level. If measured with a sufficiently high time resolution, a polyphasic transient with four distinct steps, designated as O, J, I, and P, is observed. After reaching maximum intensity at the P step, the fluorescence intensity declines until it reaches a steady state within a few minutes (Harbinson and Rosenqvist, 2003; Govindjee, 2004).The physiological mechanisms underlying the polyphasic OJIP transient are still not clarified, but it is believed that the J and I steps represent dynamic bottlenecks in the photosynthetic electron transport chain. The first rise (2 ms) from O to J is referred to as the photochemical phase due to its dependence on the intensity of the incoming light. This phase is assumed to reflect the reduction of the primary quinone electron acceptor in PSII (Stirbet and Govindjee, 2011). The reduction of the primary quinone electron acceptor results in a decreased electron trapping efficiency and therefore an increase in the dissipation of absorbed light energy by fluorescence and heat. The second part, from J over I to P, is called the thermal phase due to its temperature sensitivity. This phase is much slower than the first, and it is believed that the J-I phase primarily reflects a sequential reduction of the remaining plastoquinone pool of PSII and that the I-P phase reflects the subsequent electron flow through cytochrome b₆f to electron sinks at the PSI acceptor side (Stirbet and Govindjee, 2011). Thus, the OJIP transient resembles a titration of the photochemical quantum yield and reflects the complex electron transport properties of PSII and PSI.Consistent with their known influence on photosynthesis, deficiencies of essential plant nutrients such as Fe, Cu, Mg, Mn, and S have previously been shown to affect OJIP transients (Kastori et al., 2000; Mallick and Mohn, 2003; Larbi et al., 2004; Husted et al., 2009; Tang et al., 2012; Yang et al., 2012). As a consequence, several attempts have been made to identify nutrient imbalances and disorders using one or several parameters derived from the transients, but apart from Mn (Husted et al., 2009; Schmidt et al., 2013), attempts have not been successful in terms of sensitivity and specificity. This includes P, which previously has been reported to have an effect on OJIP transients, yet the reported effects seem mutually contradictory and nonspecific to P (Ripley et al., 2004; Weng et al., 2008; Jiang et al., 2009; Lin et al., 2009).Here, we present the unique finding that increasing levels of P deficiency affect the shape of the OJIP transient around the I step at 20 to 50 ms and causes the I step to gradually straighten and disappear. It is demonstrated that this effect is fully reversible and, among the essential plant nutrients, specific for P deficiency using both monocotyledons (barley [Hordeum vulgare]) and dicotyledons (tomato [Solanum lycopersicum]) plant species. Furthermore, it is shown that it is possible to determine whether a plant is P sufficient or deficient and to quantitatively predict the P concentration in leaf tissue using multivariate analysis of the OJIP transients.     

Rapid and Sensitive Detection of Malachite Green and Melamine with Silver Film over Nanospheres by Surface-Enhanced Raman Scattering

Plasmonics - Silver film over nanospheres (AgFON) have been prepared by depositing Ag thin film onto the self-assembled monolayer arrays of polystyrene nanospheres with vacuum magnetron sputtering...     

高灵敏焦测序结合生物发光分析仪检测甲型H1N1流感病毒

目的:建立基于核酸序列分析的快速、准确、低成本的甲型H1N1流感病毒检测方法。方法:通过优化焦测序反应体系中ATP硫酸化酶和荧光素酶的浓度,建立高灵敏的焦测序反应体系;将该体系应用于低成本、小型化的便携式生物发光分析仪,焦测序分析流感病毒M、NP、HA基因片段的核酸序列。结果:优化后的焦测序反应体系可检测低至10 fmol的DNA样本,检测灵敏度较传统焦测序提高了10倍以上。对两例样本进行检测,根据所测得的M、NP、HA基因特异性片段序列,可以确认其均为甲型H1N1感染;另外,对M2蛋白阻断剂耐药性标志位点(S31N突变)的测定结果显示该病毒存在S31N突变,为M2蛋白阻断剂耐药型。结论:高灵敏焦测序体系结合便携式生物发光分析仪成功实现了对甲型H1N1流感病毒快速、准确的低成本检测。     

Development of a Highly Sensitive Method for Detection of Clarithromycin-Resistant Helicobacter pylori from Human Feces

Gastric infection of clarithromycin (CAM)-resistant Helicobacter pylori is one of the major causes of failure to eradicate this organism. A noninvasive and useful method for the detection of CAM-resistant H. pylori from human feces by restriction fragment length polymorphism (RFLP)-nested polymerase chain reaction (PCR) targeting the mutation of the 23S rRNA gene that confers CAM-resistance in H. pylori was developed in this study. Our nested PCR method detected DNA of H. pylori in feces with high sensitivity and specificity compared with both an enzyme-linked immunoadsorbent assay (ELISA) of H. pylori in feces and the isolation of H. pylori from gastric biopsy. Furthermore, the results of mutation analysis of the H. pylori 23S rRNA gene amplified from feces completely correlated with both that of the H. pylori 23S rRNA gene amplified from the isolates of gastric biopsy and the susceptibility of H. pylori isolates to CAM. Therefore, our results show that this RFLP/nested PCR method is useful for the accurate diagnosis of CAM-resistant H. pylori infection from feces.     

Improved Detection of Magnetic Signals by a MEMS Sensor Using Stochastic Resonance

We introduce the behavior of the electrical output response of a magnetic field sensor based on microelectromechanical systems (MEMS) technology under different levels of controlled magnetic noise. We explored whether a particular level of magnetic noise applied on the vicinity of the MEMS sensor can improve the detection of subthreshold magnetic fields. We examined the increase in the signal-to-noise ratio (SNR) of such detected magnetic fields as a function of the magnetic noise intensity. The data disclosed an inverted U-like graph between the SNR and the applied magnetic noise. This finding shows that the application of an intermediate level of noise in the environment of a MEMS magnetic field sensor improves its detection capability of subthreshold signals via the stochastic resonance phenomenon.     

高通量流式荧光微球悬液芯片检测乙肝病毒基因分型

目的:建立HBV基因分型高通量液相芯片检测技术,并探讨其应用价值.方法:对GenBank中收录的明确分型的HBV基因序列进行分析,选择preS2-S区设计引物和A、B、C和D型特异性探针.与荧光编码微球偶联的特异型探针与一条引物生物素标记的PCR产物直接杂交反应,然后结合亲和素标记的藻红蛋白,用流式检测仪(Bio-Plex 200)检测荧光信号.检测182份阳性乙肝患者血清DNA,其中35份样品检测结果与测序法比较.用B、C型质粒DNA倍比稀释及混合样品检测灵敏度来评估该方法.结果:建立了HBV基因分型的快速高通量液相芯片检测方法.182份患者血清检测结果为:B型占24.2％ (44/182),C型占71.4％(130/182),D型为6.6 ％(12/182),BC混合型4.4％(8/182).其中35份样本与测序法比较,除3份混合型测序法未检出外,其它32例结果均相同本方法的灵敏度检测下线为1×103 copies/mL.结论:应用悬液芯片技术进行乙肝病毒的基因分型分析,具有较好的特异性和较高的灵敏度,并有简便、灵活和高通量等优势.该检测系统不仅在科研中有广泛的前景,也有望成为临床推广的多重分子诊断和基因分型的新方法.     

Simultaneous Detection of Fenitrothion and Chlorpyrifos-Methyl with a Photonic Suspension Array

A technique was developed for simultaneous detection of fenitrothion (FNT) and chlorpyrifos-methyl (CLT) using a photonic suspension array based on silica colloidal crystal beads (SCCBs). The SCCBs were encoded with the characteristic reflection peak originating from the stop-band of colloidal crystal. This approach avoids the bleaching, fading or potential interference seen when encoding by fluorescence. SCCBs with a nanopatterned surface had increased biomolecule binding capacity and improved stability. Under optimal conditions, the proposed suspension array allowed simultaneous detection of the selected pesticides in the ranges of 0.25 to 1024 ng/mL and 0.40 to 735.37 ng/mL, with the limits of detection (LODs) of 0.25 and 0.40 ng/mL, respectively. The suspension array was specific and had no significant cross-reactivity with other chemicals. The mean recoveries in tests in which samples were spiked with target standards were 82.35% to 109.90% with a standard deviation within 9.93% for CLT and 81.64% to 108.10% with a standard deviation within 8.82% for FNT. The proposed method shows a potentially powerful capability for fast quantitative analysis of pesticide residues.     

利用厚度剪切模传感器在线快速检测鼠伤寒沙门氏菌

鼠伤寒沙门氏菌的浓度和生长用厚度剪切模（ＴＳＭ）传感器在线进行了测定．通过实验发现该法简单、实时和快速．该传感器的响应和细胞浓度之间存在良好的线性关系,相关系数为０．９７４．细菌的生长初始期即延滞期和对数生长期可以用该装置在线测定．影响测定的诸因素也进行了讨论,如培养基和电解质是关键的因素．测定的最佳浓度范围为１．２５×１０５～１×１０１０细胞／升．