Microscale biosensor for measurement of volatile fatty acids in anoxic environments

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO(-)(3) to N(2)O. A culture of denitrifying bacteria deficient in N(2)O reductase was immobilized in front of the tip of an electrochemical N(2)O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N(2)O sensor. The signal of the sensor, which corresponded to the rate of N(2)O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O(2), N(2)O, NO(2)(-), H(2)S, and NO(-)(3). Interference from NO(-)(3) was low and could be quantified and accounted for. The detection limit was equivalent to about 1 microM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32 per thousand. The functioning of the sensor over a temperature span of 7 to 30 degrees C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5 degrees C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.     

Rapid and sensitive biosensor for Salmonella

The rapid and sensitive detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated. The binding of bacteria to the surface changed the crystal resonance parameters; these were quantified by the output voltage of the sensor instrumentation. The sensor had a lower detection limit of a few hundred cells/ml, and a response time of < 100 s over the range of 10(2)-10(10) cells/ml. The sensor response was linear between bacterial concentrations of 10(2)-10(7) cells/ml, with a sensitivity of 18 mV/decade. The binding of bacteria was specific with two binding sites needed to bind a single cell. The sensors preserve approximately 75% of their sensitivity over a period of 32 days.     

Ex vivo measurement of lumbar intervertebral disc pressure using fibre-Bragg gratings

Methods were developed to measure intervertebral disc pressure using optical fibre-Bragg gratings (FBGs). The FBG sensor was calibrated for hydrostatic pressure in a purpose-built apparatus and the average sensitivity was determined to be -5.7 +/- 0.085 pm/MPa (mean +/- SD). The average coefficient of determination (r(2)) for the calibration data was 0.99, and the average hysteresis of the sensor was 2.13% of full scale. The FBG was used to measure intradiscal pressure response to compressive load in five lumbar functional spine units. The pressure measured by the FBG sensor varied linearly with applied compressive load with coefficients of determination ranging from 0.84 to 0.97. The FBG sensor's sensitivity to compressive load ranged from 0.702 +/- 0.043 kPa/N (mean +/- SD) in a L1-L2 specimen, to 1.07 +/- 0.069 kPa/N in a L4-L5 specimen. These measurements agree with those of previous studies in lumbar spines. Two strain gauge pressure sensors were also used to measure intradiscal pressure response to compressive load. The measured pressure sensitivity to load ranged from 0.251 kPa/N (L4-L5) to 0.850 kPa/N (L2-L3). The average difference in pressure sensitivity to load between Sensors 1 and 2 was 12.9% of the value for Sensor 1, with a range from 1.1% to 20.4%, which suggests that disc pressure was not purely hydrostatic. This may have contributed to the difference between the responses of the FBG and strain gauge sensors.     

Surface functionalization allowing repetitive use of optical sensors for real‐time detection of antibody‐bacteria interaction

In this study, sensor surface functionalization allowing the repetitive use of a sensing device was evaluated for antibody‐based detection of living bacteria using an optical planar Bragg grating sensor. To achieve regenerable immobilization of bacteria specific antibodies, the heterobifunctional cross‐linker N‐succinimidyl 3‐(2‐pyridyldithio) propionate (SPDP) was linked to an aminosilanized sensor surface and subsequently reduced to expose sulfhydryl groups enabling the covalent conjugation of SPDP‐activated antibodies via disulfide bonds. The immobilization of a capture antibody specific for Staphylococcus aureus on the sensor surface as well as specific binding of S. aureus could be monitored, highlighting the applicability of optical sensors for the specific detection of large biological structures. Reusability of bacteria saturated sensors was successfully demonstrated by cleaving the antibody along with bound bacteria through reduction of disulfide bonds and subsequent re‐functionalization with activated antibody, resulting in comparable sensitivity towards S. aureus.

     

酶电极流动注射分析法测定葡萄糖

 一种酶电极流动注射分析系统(EFIA)用于血糖和发酵葡萄糖的快速测定。研究了酶电极及其工作系统的性能和各种影响参数,,奠定了实用化基础。     

Palladium hexacyanoferrate hydrogel as a novel and simple enzyme immobilization matrix for amperometric biosensors

An amperometric glucose biosensor with glucose oxidase (GOx) immobilized into palladium hexacyanoferrate (PdHCF) hydrogel has been prepared and evaluated. The sensor was based on a two-layer configuration with biocatalytic and electrocatalytic layers separately deposited onto the electrode. To reduce the overpotential for reduction of hydrogen peroxide liberated in the enzyme catalyzed oxidation of glucose, an inner thin layer of nickel hexacyanoferrate (NiHCF) electrodeposited onto the surface of graphite electrode was used as an electrocatalyst. As an outer layer, the hydrogel of palladium hexacyanoferrate with entrapped glucose oxidase was used. Under optimal operating conditions (pH 5.0 and E = -0.075 V versus calomel (3.0 M KCl) reference electrode), sensor showed high sensitivity to glucose (0.3-1.0 microA/mM) and a response time of less than 30s. The linear response to glucose was obtained in the concentration range between 0.05 and 1.0 mM in batch analysis mode and 0-7.0 mM in FIA. During the 32 days testing period, no significant decrease in the sensor sensitivity was observed. The sensor was applied for the determination of glucose concentration in fruit juice and yoghurt drink, and the results obtained showed good correlation with results obtained by reference spectrophotometric enzyme method.     

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.     

Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved‐core alginate microspheres

The feasibility of dissolved‐core alginate‐templated fluorescent microspheres as “smart tattoo” glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye‐labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution‐phase counterparts. The glucose sensitivity of the encapsulated TRITC‐Con A/FITC‐dextran (500 kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC‐apo‐GOx/FITC‐dextran (500 kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0–50 mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120 s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0–50 mM glucose, using near‐infrared dyes (Alexa Fluor‐647‐labeled dextran as donor and QSY‐21‐conjugated apo‐GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075–1085. © 2009 Wiley Periodicals, Inc.     

A Microsensor for Nitrate Based on Immobilized Denitrifying Bacteria

A biosensor for NO(inf3)(sup-) was constructed by attaching a 30- to 70-(mu)m-wide capillary with immobilized denitrifying bacteria in front of an N(inf2)O microsensor. These bacteria reduced O(inf2) so that only bacteria in the very tip of the sensor were exposed to O(inf2) whereas bacteria at a greater depth could carry out the anaerobic process of denitrification. In the presence of acetylene, which inhibits nitrous oxide reductase, bacteria reduced NO(inf3)(sup-) (or NO(inf2)(sup-)) from the surrounding medium to N(inf2)O and the concentration sensed by the N(inf2)O microsensor was directly proportional to the concentration of NO(inf3)(sup-) in the medium. By applying a 250-(mu)m-long capillary in front of the N(inf2)O microsensor, the 90% response time of the biosensor was 50 s. Biosensors may also be made with nitrous oxide-deficient strains so that acetylene inhibition can be omitted.     

Bioavailable nitrate detection in water by an immobilized luminescent cyanobacterial reporter strain

Cyanobacteria are a major group of photosynthetic bacteria that can accumulate in surface water as so-called "blooms" in response to environmental factors such as temperature, light and certain nutrients such as N, P, and Fe. Some species of cyanobacteria produce toxins, causing a considerable danger for human and livestock health. As a consequence, monitoring of bloom formation and toxin production of drinking water supplies has become a major concern. To enable prediction and monitoring of cyanobacterial blooms, tools to detect nutrient bioavailability in water would be advantageous. A whole-cell biosensor was developed for monitoring nitrate (NO(3-)) bioavailability in aquatic ecosystems using the recombinant bioluminescent cyanobacterial strain Synechocystis PCC 6803 harboring an insertion of a luxAB-kmr fusion with nblA1 in its chromosomal DNA, leading to PnblA::luxAB-kmr. This reporter strain was designated N1LuxKm. Cells were immobilized in microtiter plates and showed a dose-dependent response to nitrate deprivation. The resultant CyanoSensor could detect nitrate in the 4-100 micro M concentration range after a sample incubation time of 10 h under continuous illumination (50 micro E m(-2) s(-1)). The optimal temperature for sensor operation was 29 degrees C and the immobilized biosensor could be stored at 4 degrees C in dark for about 1 month without significant loss of sensitivity.     

Electrochemical sensor based on gold nanoparticles fabricated molecularly imprinted polymer film at chitosan-platinum nanoparticles/graphene-gold nanoparticles double nanocomposites modified electrode for detection of erythromycin

A molecularly imprinted electrochemical sensor was fabricated based on gold electrode decorated by chitosan-platinum nanoparticles (CS-PtNPs) and graphene-gold nanoparticles (GR-AuNPs) nanocomposites for convenient and sensitive determination of erythromycin. The synergistic effects of CS-PtNPs and GR-AuNPs nanocomposites improved the electrochemical response and the sensitivity of the sensor. The molecularly imprinted polymers (MIPs) were prepared by HAuCl(4), 2-mercaptonicotinic acid (MNA) and erythromycin. Erythromycin and MNA were used as template molecule and functional monomer, respectively. They were first assembled on the surface of GR-AuNPs/CS-PtNPs/gold electrode by the formation of Au-S bonds and hydrogen-bonding interactions. Then the MIPs were formed by electropolymerization of HAuCl(4), MNA and erythromycin. The sensor was characterized by cyclic voltammetry (CV), scanning electron microscope (SEM), UV-visible (UV-vis) absorption speactra and amperometry. The linear range of the sensor was from 7.0×10(-8)mol/L-9.0×10(-5)mol/L, with the limit of detection (LOD) of 2.3×10(-8)mol/L (S/N=3). The sensor showed high selectivity, excellent stability and good reproducibility for the determination of erythromycin, and it was successfully applied to the detection of erythromycin in real spiked samples.     

Evaluation of a new fiber-optic pressure recording system for cardiovascular measurements in mice

We have tested a new fiber-optic pressure recording system, Samba, with a thin fiber [outer diameter (OD) = 0.25 mm] and a pressure sensor (length and OD = 0.42 mm) attached to the end. The accuracy of the system tested in vitro was good, with a coefficient of variation of 2.54% at 100 mmHg. The drift was <0.45 mmHg/h, and the temperature sensitivity was approximately 0.07 mmHg/1 degrees C between 22 and 37 degrees C. The frequency response characteristics were similar to a 1.4-Fr Millar catheter (0-200 Hz). Introduction of the Samba sensor from the right carotid artery into the left ventricle in six mice caused no drop in mean aortic pressure, whereas introduction of a 1.4-Fr Millar catheter (OD = 0.47 mm; n = 6) caused a pressure drop from 91.6 +/- 9.2 to 65.1 +/- 6.2 mmHg; P < 0.05. Thus the Samba sensor system may represent a new alternative to assess hemodynamic variables in the murine cardiovascular system.     

Sheep manure vermicompost supplemented with a native diazotrophic bacteria and mycorrhizas for maize cultivation

An orthogonal experimental design L9 (3(4)) with 10 repetitions was used to investigate the effect of Glomus claroideum (0, 1 or 2g(-1) plant), G. fasciculatum (0, 1 or 2g plant(-1)), native diazotrophic bacteria (0, 10(3) and 10(5) UFC ml(-1)) and sheep manure vermicompost (0%, 5% and 10% v/v) on maize plant growth, N and P in leaves and mycorrhization percent. Vermicompost explained most of the variation found for leaf number, wet weight, stem height, and diameter. Both mycorrhizas increased the plant wet weight but G. fasciculatum the most. Mycorrhization increased the P content, but not the N content. Mycorrhizal colonization increased when diazotrophic bacteria and vermicompost were added. It was found that weight of maize plants cultivated in peat moss amended with vermicompost increased when supplemented with G. fasciculatum and diazotrophic bacteria.     

Fabrication of an oxytetracycline molecular-imprinted sensor based on the competition reaction via a GOD-enzymatic amplifier

A highly sensitive molecular-imprinted polymer sensor (MIP sensor) for ultratrace oxytetracycline (OTC) determination was prepared based on the competition reaction between template molecule OTC and glucose oxidase (GOD)-labeled OTC (GOD-OTC). Sensitivity improved dramatically due to the detection of a huge amount of enzyme catalytic production, which was inversely proportional to template molecule concentration. The MIP sensor was characterized by alternating current impedance spectroscopy and cyclic voltammetry, and its voltammetric behavior was also verified. Experimental conditions including isolation, incubation, and competition were optimized. OTC can be determined at concentrations between 0 and 4.0×10(-7) mol/L with a detection limit of 3.30×10(-10) mol/L by the differential pulse voltammetry technique. The MIP sensor showed high sensitivity, selectivity, reproducibility, and good recovery in sample determination.     

Microscale Biosensor for Measurement of Volatile Fatty Acids in Anoxic Environments

A microscale biosensor for acetate, propionate, isobutyrate, and lactate is described. The sensor is based on the bacterial respiration of low-molecular-weight, negatively charged species with a concomitant reduction of NO₃⁻ to N₂O. A culture of denitrifying bacteria deficient in N₂O reductase was immobilized in front of the tip of an electrochemical N₂O microsensor. The bacteria were separated from the outside environment by an ion-permeable membrane and supplied with nutrients (except for electron donors) from a medium reservoir behind the N₂O sensor. The signal of the sensor, which corresponded to the rate of N₂O production, was proportional to the supply of the electron donor to the bacterial mass. The selectivity for volatile fatty acids compared to other organic compounds was increased by selectively enhancing the transport of negatively charged compounds into the sensor by electrophoretic migration (electrophoretic sensitivity control). The sensor was susceptible to interference from O₂, N₂O, NO₂⁻, H₂S, and NO₃⁻. Interference from NO₃⁻ was low and could be quantified and accounted for. The detection limit was equivalent to about 1 μM acetate, and the 90% response time was 30 to 90 s. The response of the sensor was not affected by changes in pH between 5.5 and 9 and was also unaffected by changes in salinity in the range of 2 to 32‰. The functioning of the sensor over a temperature span of 7 to 30°C was investigated. The concentration range for a linear response was increased five times by increasing the temperature from 7 to 19.5°C. The life span of the biosensor varied between 1 and 3 weeks after manufacturing.     

华西雨屏区天然常绿阔叶林土壤可培养微生物数量对模拟氮沉降的响应

为了解氮沉降对华西雨屏区天然常绿阔叶林土壤微生物数量的影响,从2013年11月至2014年12月,通过野外模拟N(NH_4NO_3)沉降,氮沉降水平分别为对照(CK 0 kg N hm~(-2)a~(-1))、低氮沉降(L 50 kg N hm~(-2)a~(-1))、中氮沉降(M 150 kg N hm~(-2)a~(-1))和高氮沉降(H 300 kg N hm~(-2)a~(-1)),研究了氮沉降对天然常绿阔叶林0—10cm和10—20cm土层土壤可培养微生物数量的影响。结果表明:华西雨屏区天然常绿阔叶林0—10cm土层的细菌、真菌和放线菌数量均显著大于10—20cm土层,氮沉降未改变原有垂直分布格局。L处理对0—10cm和10—20 cm土层土壤微生物总量无显著影响,M和H处理则显著降低了土壤微生物总量。氮沉降降低了0—10cm和10—20cm土层的细菌数量,且抑制作用随氮沉降量的增加而增强。氮沉降降低了0—10cm土层的真菌数量,但下降幅度与氮沉降量之间无明显规律;在10—20cm土层,M和H处理在夏季显著增加了真菌数量,表明适量氮沉降能有效缓解夏季土壤真菌的氮限制状态。氮沉降对0—10cm土层放线菌数量的影响表现为先促进再抑制,L和M处理增加了放线菌数量,H处理降低了放线菌数量;氮沉降增加了10—20cm土层的放线菌数量,其中M处理的促进作用最大。氮沉降对土壤微生物数量的影响随土壤深度的增加而减弱。     

A novel immunosensor based on an alternate strategy of electrodeposition and self-assembly

An imprinted electrochemical sensor based on polypyrrole-sulfonated graphene (PPy-SG)/hyaluronic acid-multiwalled carbon nanotubes (HA-MWCNTs) for sensitive detection of tryptamine was presented. Molecularly imprinted polymers (MIPs) were synthesized by electropolymerization using tryptamine as the template, and para-aminobenzoic acid (pABA) as the monomer. The surface feature of the modified electrode was characterized by cyclic voltammetry (CV). The proposed sensor was tested by chronoamperometry. Several important parameters controlling the performance of the molecularly imprinted sensor were investigated and optimized. The results showed that the PPy-SG composites films showed improved conductivity and electrochemical performances. HA-MWCNTs bionanocomposites could enhance the current response evidently. The good selectivity of the sensor allowed three discriminations of tryptamine from interferents, which include tyramine, dopamine and tryptophan. Under the optimal conditions, a linear ranging from 9.0×10(-8) mol L(-1) to 7.0×10(-5) mol L(-1) for the detection of tryptamine was observed with the detection limit of 7.4×10(-8) mol L(-1) (S/N=3). This imprinted electrochemical sensor was successfully employed to detect tryptamine in real samples.     

土壤微生物数量对模拟氮沉降增加的早期响应

通过以南亚热带森林三种主要树种即荷木、锥栗和黄果厚壳桂为主的苗圃试验地的苗圃控制实验,初步探讨土壤微生物数量对模拟氮沉降增加的响应。结果表明,施氮增加对土壤微生物数量的影响根据类群和氮处理水平不同而异。总的来说,施氮增加对土壤微生物数量具有促进作用,这种促进作用对放线菌数量仅在一定(中氮)处理水平以下,超过此水平施氮增加则表现为抑制作用,而施氮对真菌数量则始终表现为抑制作用,尤其以中N处理水平的抑制作用最强。但目前细菌仍占微生物总量的绝对优势,放线菌次之,真菌则占微生物总量的比例最小。分析结果还表明,有效氮与放线菌、有效氮与细菌数量变化呈显著相关关系。     

A magnetoelastic resonance biosensor immobilized with polyclonal antibody for the detection of Salmonella typhimurium

Mass-sensitive, magnetoelastic resonance sensors have a characteristic resonant frequency that can be determined by monitoring the magnetic flux emitted by the sensor in response to an applied, time varying, magnetic field. This magnetostrictive platform has a unique advantage over conventional sensor platforms in that measurement is wireless and remote. A biosensor for the detection of Salmonella typhimurium was constructed by immobilizing a polyclonal antibody (the bio-molecular recognition element) onto the surface of a magnetostrictive platform. The biosensor was then exposed to solutions containing S. typhimurium bacteria. Binding between the antibody and antigen (bacteria) occurred and the additional mass of the bound bacteria caused a shift in the sensor's resonant frequency. Sensors with different physical dimensions were exposed to different concentrations of S. typhimurium ranging from 10(2) to 10(9)CFU/ml. Detection limits of 5x10(3) CFU/ml, 10(5) CFU/ml and 10(7) CFU/ml were obtained for sensors with the size of 2 mmx0.4 mmx15 microm, 5 mmx1 mmx15 microm and 25 mmx5 mmx15 microm, respectively. Good agreement between the measured number of bound bacterial cells (as measured by scanning electron microscopy (SEM)) and frequency shifts was obtained.     

种植密度与施氮量对麦茬复种饲料油菜土壤微生物活性的影响

通过田间小区试验,研究了不同油菜种植密度与施氮肥对麦茬复种饲料油菜耕层土壤(0～5 cm)微生物活性的影响.结果表明,复种油菜能显著或极显著性提高耕层土壤微生物量碳(C_mic)、土壤微生物量氮(N_mic)、土壤细菌数(SBN)、土壤真菌数(SFN)和土壤放线菌数(SAN),而显著降低土壤微生物量碳/氮比(C_mic/N_mic).随油菜种植密度的提高,耕层C_mic、C_mic/N_mic、SBN呈逐渐增加态势,而N_mic、SAN呈降低趋势.随着施氮肥梯度增加,耕层SFN显著提高,C_mic和N_mic呈先降低后增加再降低趋势,以1 000 kg·hm^-2施肥处理最高.SFN和收获期SAN为先降低后升高,苗期SAN则为先升高后降低.相同处理油菜收获期各土壤微生物活性指标均高于苗期,而600 kg·hm^-2苗期SAN则相反.SBN和SAN与C_mic、N_mic呈正相关,与C_mic/N_mic呈负相关,SFN与C_mic、N_mic及C_mic/N_mic之间均无明显相关性.