We report a method for efficient mutagenesis of DNA in large vectors without subcloning. Two segments of the target DNA sequence, one having a mutation introduced via a mutant primer, were amplified by PCR and then the purified fragments were ligated to a vector. The mutation efficiency was nearly 100%.

Firmness is an important quality indicator of kiwifruit and is useful for determining optimal time for marketing and optimizing storage management. In this study, a self-designed system based on acoustic vibration technology was used to realize real-time detection of kiwifruit firmness. To ensure stability of measurement, impact force of the excitation device in the system was calibrated to 12.03 +/- 0.71 N. The acoustic vibration response signals of kiwifruit were converted from time domain to frequency domain and 10 statistical features were extracted. Most of the features had good correlations with reference firmness, which proved feasibility of firmness prediction using signals collected in the self-designed system. Subsequently, PLS regression models for predicting firmness were established based on frequency-domain spectra. To further improve accuracy of the model, CARS algorithm was used to select effective frequencies that were highly correlated with kiwifruit firmness. According to the results, prediction accuracy of the CARS-PLS model in external cross-validation sets for flesh firmness was the best (R-cv(2) = 0.96, RMSECV = 0.27, and RPDcv = 5.21), followed by stiffness (R-cv(2) = 0.95, RMSECV = 0.43, and RPDcv = 5.00), and prediction accuracy of the model for skin firmness was the worst (R-cv(2) = 0.93, RMSECV = 0.81, and RPDcv = 4.01). Overall, acoustic vibration signals obtained by the self-designed device in a nondestructive way can well characterize the firmness of kiwifruit. The proposed method in this study can achieve high-precision prediction of all three kiwifruit firmness indices and meet requirements of online real-time detection.

Exploring new functions of nanomaterials can help facilitate the development of biosensors for the detection of antibiotics. Herein, a new detection modality based on monovalent antigen-induced aggregation (MAA) of immunomagnetic beads (IMBs) was proposed for rapid and label-free detection of enrofloxacin (ENR), which endowed IMBs with the abilities of both sample separation and signal generation. In the presence of ENR, the initially well-dispersed IMBs were aggregated and the degree of aggregation was in a concentration-dependent manner. After exploring the mechanism underlying IMB aggregation and investigating the key parameters affecting it, a label-free biosensing platform was developed for rapid and sensitive detection of ENR. Based on the significant differences in the magnetic separation speed and size between the aggregated and well-dispersed IMBs, two methods were proposed for quantitatively determining ENR, i.e., measuring the turbidity of the IMB supernatant after magnetic separation for a given time and visualizing and calculating the grayscale value of the aggregated IMBs trapped on the surface of a nitrocellulose membrane. A three-dimensional (3D)-printed syringe was designed and fabricated for automatic filtration of IMBs. This immunosensor allowed for sensitive detection of ENR in less than 15 min without any labels. It exhibited a satisfactory limit of detection of 0.79 ng mL-1 and showed the feasibility for ENR detection of spiked chicken meat with recovery rates ranging from 74.8 to 98.3%. The MAA immunosensor can act as a promising tool to detect trace levels of ENR and has the potential to be applied to complex food samples.

HighlightsThe impact sensors protected by shells and integrated with process circuits were proposed.Simulations and collision tests were performed to optimize the structure of the sensor.Accessories that can improve sensors’ detection accuracy were proposed.Multi-grain impact tests in a laboratory showed the RMSEs of two sensors were less than 1.67%.Abstract. Impact sensors are widely used to detect grain loss in harvesters, and the impact sensors with array structure polyvinylidene fluoride (PVDF) films have been proven to significantly increase the detection efficiency. However, uneven sensitivity and clutters from non-impacted sensitive units seriously affect the detection accuracy. This article proposes a protective shell for the impact sensors with array structure PVDF films. To improve the performance of this kind of sensor, we optimized the structure through finite element simulations and particle impact tests. The vibration modes under three pasting methods of sensitive units were analyzed. The relationship between the hardness of the elastic layer and response performances was investigated. To suppress the clutters, a method of splitting the elastic layer was proposed. Then, we manufactured two optimized impact sensors with one layer and two crossed layers of PVDF films and tested their performances. The results demonstrated that the sensitivities of the gaps between the PVDF films are poor. But when excluding the gaps’ data, the two sensors can meet the detection requirement. Accessories were proposed based on that characteristic to prevent particles from colliding with the gaps. After assembling the accessory, multi-grain impact tests showed that the root-mean-square errors (RMSEs) of the impact sensors with one layer and two crossed layers of PVDF films were 1.67% and 1.63%, respectively. Keywords: Grain loss detection, Particle impact test, Protective shell, Sensor accessory, Structure optimization.

Some veterinary drug residues in food products and environment have been widely regarded as severe threats to human health. Rapid and simultaneous detection methods are crucial to monitor and control veterinary drug usage. Here, we propose a fluorescence biosensor utilizing immunomagnetic beads (IMBs) and quantum dots (QDs) for the rapid and simultaneous detection of 1-adamantylamine (ADA), enrofloxacin (ENR) and tilmicosin (TIL) in raw chicken meat. A pretreatment method using sodium phosphotungstate–magnesium as extraction reagent was developed to simultaneously extract ADA, ENR and TIL from chicken meat with minor interference in background or response. By adding the IMBs modified with three types of antibodies and the QD-antigens modified with three types of BSA-antigens to sample, IMBs competitively conjugated to target antigens in a sample or QD-antigens. After magnetic separation, the residual QD-antigens were adopted to collect signals using fluorescence spectroscopy. Using QDs with well separated emission peaks, the detection of one type of targets was minorly interfered by the others. Under the optimum conditions, the biosensor exhibited the limit of detection of 0.96, 3.32, and 3.17 ng/mL for ADA, ENR and TIL in chicken samples, respectively, as well as good specificity. Due to the way of direct collection of signals in extracts, the tedious and complicated multiple magnetic separation and signal amplification procedures in conventional methods were avoided, thus the procedures were significantly simplified, and the reduction of the operation time of 30 min for sample pretreatment and 40 min for detection part was achieved. The biosensor might be promising in the rapid, in-field and sensitive screening of multiple veterinary drugs to ensure agriculture and food safety.

Highlights The TS-PEF diminished the color change compared to individual treatments. The concentration of Fe, Cr, and Ni increased after the treatments of PEF and TS-PEF. The volatile flavor compounds decreased after the treatment of TS and TS-PEF. Abstract . Chinese rice wine (CRW) is a traditional wine prevalent in East Asia. In current industrial production of bottled CRW, an additional thermal pasteurization is required at the bottling stage to extend the shelf life of the product, and this thermal treatment results in quality degradation and even carcinogen formation. In our previous studies, non-thermal technologies such as pulsed electric fields (PEF), thermosonication (TS), and their combination (TS-PEF) have been shown to significantly inactivate Saccharomyces cerevisiae in CRW as an alternative to thermal pasteurization of bottled wine. However, studies of their effects on the quality of CRW are very limited. In this study, we compared the physicochemical properties of CRW treated by pasteurization, PEF, TS, and TS-PEF. The results showed that the basic physicochemical indicators of CRW (total sugar, non-sugar solids, total acidity, amino acid nitrogen, and pH) did not change significantly after all these treatments. The TS-PEF treatment reduced the total color difference caused by the TS or PEF treatments. However, corrosion of the electrodes during PEF and TS-PEF treatment caused a significant increase in iron, chromium, and nickel concentrations. In addition, volatile flavor compounds, such as alcohols, esters, acids, and aldehydes, were reduced much more after TS and TS-PEF treatment than after pasteurization and PEF. Further research is needed on minimizing the negative impacts of these new technologies on the flavor of CRW in the context of effective sterilization. Keywords: Chinese rice wine, Metal ion, Physicochemical properties, Pulsed electric fields, Ultrasound, Volatile flavor compounds.

HighlightsA practical magnetic separation device was designed, fabricated, and evaluated for enrofloxacin detection.Coupled with a fluorescent biosensor, the device could automatically process a sample in 50 min.The device performed incubation and magnetic separation using a pipette method.The device has the advantages of low-cost and feasibility for on-site detection.Abstract. Antibiotic residues have been a continuing concern in food safety, raising a great issue in human health. For rapid detection of antibiotics, an automated device was developed that can capture and separate a target analyte based on immunomagnetic beads. This automated separation device is suitable for separating the magnetic beads in a preprocessing step, with liquid transfer and magnetic enrichment functions. The device was combined with a fluorescent biosensor to simplify the cumbersome pretreatment of enrofloxacin. In our experiments, enrofloxacin in water samples was used as the detection object, and the entire process could be completed in less than 50 min with automated operation. The lower limit of detection reached 54 ng mL-1 (S/N = 3). The fluorescent biosensor has been enhanced with this automated separation device for more sensitive rapid detection of antibiotic residues in the food supply chain and environment. Keywords: Antibiotic detection, Automation, Fluorescent biosensor, Immunomagnetic separation, Sample pretreatment.

Highlights A nanobiosensor was developed for rapid detection of enrofloxacin residues in chicken meat. 5-Sulfosalicylic acid was adopted in a facile method for pretreatment of chicken meat samples. The detection limit of 14.1 µg kg -1 was below the maximum residue limit for chicken meat. The total detection time from sample pretreatment to result report was less than 1.5 h. Abstract . Antibiotic residues in animal-derived food products have been identified as a potential hazard in human health. Hence, a rapid, simple, and cost-effective method for detection of antibiotics in the food supply chain is highly desirable. The objective of this study was to develop a nanomaterial-based biosensor using immunomagnetic beads (IMBs) and quantum dots (QDs) for rapid and sensitive detection of enrofloxacin (ENR) residues in raw chicken. A 5-sulfosalicylic acid-based pretreatment method was adopted to extract ENR from chicken meat and reduce non-specific adsorption caused by complex food matrices. Two sensing elements were designed and fabricated: antibody functionalized IMBs and ENR-bovine serum albumin (BSA) conjugates modified QDs (QDs-BSA-ENR). Target ENR in samples was first captured and separated by IMBs, and then QDs-BSA-ENR, serving as a competitor and detection probe, was used to react with the residual binding sites on the IMB surfaces. With the presence of captured ENR, the binding of QDs-BSA-ENR to IMBs was competitively inhibited. Finally, the fluorescence intensity of reporting QDs in the QDs-BSA-ENR-IMBs complex at a wavelength of 614 nm was measured for the quantitation of target antibiotics. Under the optimum conditions, the proposed method allowed sensitive detection of ENR in a linear range from 1 to 100 ng mL-1 with a limit of detection (LOD) of 0.94 ng mL-1. The LOD for spiked chicken meat was 14.1 µg kg-1, which was below the maximum residue limits (MRLs) regulated in China and the European Union. The whole analytical procedure from food sampling to result report could be finished in less than 1.5 h. This nanobiosensor showed high potential for rapid and low-cost detection of ENR residues in the poultry supply chain to enhance food safety. Keywords: Enrofloxacin, Immunomagnetic beads, Nanobiosensor, Poultry, Quantum dots, Rapid detection.

Impact sensors are widely used to detect grain losses in a harvester. This work reports the protection of impact sensors with PVDF films and the optimization of various experimental parameters to maximize their performance in collision detection applications. A detailed study is carried out to experimentally optimize the method of pasting polyvinylidene fluoride (PVDF) films, as well as the material and the shape of the damping layer. Experimental tests on two types of impact sensors demonstrate that, excluding the gap positions, the sensor with one layer of PVDF films can meet the working requirements, and the impact sensor with two layers of crossed PVDF films has the potential to detect extremely dense collisions.

食源性内生孢子灭菌是当前食品非热加工领域的一大难题.针对这一难题,本文提出了一种超声波辅助的高压脉冲电场灭菌工艺,旨在寻找一种有效控制食品中内生孢子的非热加工方式.总结了国内外高压脉冲电场和超声波处理孢子的相关研究和联合灭菌工艺的研究现状,单独的超声波和超声波处理均很难达到理想的灭菌效果,最大灭菌效率为1～2个对数级.因此,超声波辅助的高压脉冲电场非热灭菌工艺应用于孢子具有乐观的应用前景.有望解决非热加工领域孢子的难题.

Impact sensors are widely used to detect grain losses in harvesters. Using polyvinylidene fluoride (PVDF) films as sensing elements is a promising way to improve sensor performance due to their high sensitivity, stability, and flexibility. However, the overlap of collision signals significantly reduces the accuracy of a sensor. To solve this problem, a novel impact sensor with two crossed PVDF films was designed and investigated. This sensor has two orthogonal layers of sensing elements that both respond to impacts, which creates positioning information for the impacts. Because of the sensor’s structure, a signal processing method was designed based on multisensor fusion theory. Tests were performed to verify the performance of the proposed impact sensor. The average signal-to-noise ratios (SNRs) for impacted PVDF films were 34.79 and 20.23 dB, respectively, for the upper and lower layers, while the average signal-to-clutter ratios (SCRs) for nonimpacted films were 21.90 and 10.05 dB, respectively. The sensor also has an extremely high detection efficiency of at least 1528 collisions per second and can identify particles that impact at the same time. Keywords: Grain loss detection, Impact sensors, Multisensor fusion, Particle impact tests, PVDF films.

The dynamic weighing system is used to measure the weight of eggs in motion for automatic quality inspection and grading. It is difficult to obtain the precious weight of rolling poultry eggs online from the weighing rail in a short time, due to disturbances generated by different rolling state and speed as well as the egg mass and shape. An innovative mass estimator which is suitable for embedded devices was proposed to process weighing signals online. This research presented a sorting-based mass estimator (SME) that is consisted of a finite impulse response (FIR) digital filter and asymmetrically trimmed mean. In this estimator, the FIR filter was designed for removing high-frequency disturbances, and the median was used as a reference to intercept the stationary signal from a sorted sequence of weighing signal. To make algorithms work on the online dynamic weighing equipment, an data acquisition and processing platform with capabilities of data acquisition, advanced signal processing, and pulsed signal triggering based on the Digital Signal Processor (DSP) was designed and implemented. Furthermore, a C#-based software was developed to save weighing data and calculate parameters of algorithms. The results demonstrated that almost all of the egg weighing errors were less than 1g with different processing speed (3~5 eggs s-1).

It was recently shown that electrolysis may play a substantial detrimental role in microfluidic electroporation. To overcome this problem, we have developed a non-electrolytic micro/nano electroporation (NEME) electrode surface, in which the metal electrodes are coated with a dielectric. A COMSOL based numerical scheme was used to simultaneously calculate the excitation frequency and dielectric material properties dependent electric field delivered across the dielectric, fluid flow, electroporation field and Clausius-Mossotti factor for yeast and E. coli cells flowing in a channel flow across a NEME surface. A two-layer model for yeast and a three-layer model for E. coli was used. The numerical analysis shows that in NEME electroporation, the electric fields could induce electroporation and dielectrophoresis simultaneously. The simultaneous occurrence of electroporation and dielectrophoresis gives rise to several interesting phenomena. For example, we found that a certain frequency exists for which an intact yeast cell is drawn to the NEME electrode, and once electroporated, the yeast cell is pushed back in the bulk fluid. The results suggest that developing electroporation technologies that combine, simultaneously, electroporation and dielectrophoresis could lead to new applications. Obviously, this is an early stage numerical study and much more theoretical and experimental research is needed.

Antibiotic residues in animal-derived food products have been identified as a potential hazard in human health. Hence, a rapid, simple and cost-effective method for antibiotic detection in food supply chain is highly desirable. The objective of this study was to develop a nanomaterials-based biosensor using immunomagnetic beads (IMBs) and quantum dots (QDs) for rapid and sensitive detection of enrofloxacin (ENR) in chicken products. A 5-sulfosalicylic acid-based pretreatment method was adopted to reduce non-specific adsorption caused by complex food matrices. Two sensing elements, antibody functionalized magnetic beads and enrofloxacin-bovine serum albumin conjugates modified QDs (QDs-BSA-ENR) were designed and fabricated. Target ENR in samples was first captured and separated by IMBs. Then, QDs-BSA-ENR, served as a competitor and detection probe, was used to react with the residual binding sites on IMBs surface. With the presence of captured ENR, the binding of QDs-BSA-ENR to IMBs surface was competitively inhibited. Finally, the fluorescence of all reporting QDs in the QDs-BSA-ENR-IMBs complex was measured for the quantitation of target antibiotics. Under the optimum conditions, the proposed method allowed for sensitive detection of ENR in a linear range from 1 to 100 ng mL-1 with a limit of detection (LOD) of 0.94 ng mL-1. The LOD for spiked chicken muscle samples was 16.26 μg kg-1, which was below the maximum residue limits regulated in China and the European Union. The whole analytical procedure from food sampling to result report could be finished within 1 h. This nanobiosensor showed high potential for rapid, low-cost and in-field detection of ENR in poultry supply chain to enhance food safety.

Micro and nano technologies are of increasing importance in microfluidics devices used for electroporation (electroporation – the permeabilization of the cell membrane with brief high electric field pulses). Electrochemical reactions of electrolysis occur whenever an electric current flows between an electrode and an ionic solution. It can have substantial detrimental effects, both on the cells and solutions during the electroporation. As electrolysis is a surface phenomenon, between electrodes and solution, the extent of electrolysis is increased in micro and nano electroporation over macro-electroporation, because the surface area of the electrodes in micro and nano electroporation is much larger. A possible way to eliminate the electrolytic effect is to develop non-electrolytic microelectroporation by coating the microelectroporation devices with a dielectric insulating layer. In this study, we examine the effect of a dielectric insulating layer on the performance of a singularity microelectroporation device that we have recently designed. Using numerical analysis, we study the effects of various design parameters including, input sinusoidal voltage amplitude and frequency, geometrical configuration and material electrical properties on the electroporation performance of the non-electrolytic microelectroporation device. In the simulation, we used properties of four real dielectric materials and four solutions of interest for microelectroporation. We characterized the effect of various design parameters of relevance to singularity based microelectroporation, on non-electrolytic microelectroporation. Interestingly, we found that the system behaves in some aspects as a filter and in many circumstances saturation of performance is reached. After saturation is reached, changes in parameters will not affect the performance of the device.

Plasma-activated water (PAW) is a promising nonthermal technology in food preservation and food safety. The inactivation efficacy of PAW was investigated against Saccharomyces cerevisiae CICC 1374 inoculated on grape berries. PAW30 and PAW60 were obtained by activating water with plasma for 30 and 60 min, respectively. Grapes were directly treated with PAW, and a 0.38- to 0.53-log CFU/ml reduction of S. cerevisiae was achieved in a time-dependent manner (P < 0.05). The oxidation-reduction potential and pH values of PAW30 and PAW60 were also in a time-dependent manner (P < 0.05). Grape quality assessment demonstrated no significant change in surface color and total anthocyanin content after 30 min of PAW60 treatment (P > 0.05). Although grape quality was unaffected by PAW in this study, this technology should be optimized to enhance inactivation efficiency.

A simple, highly-automated instrument system used for on-site detection of foodborne pathogens based on fluorescence was designed, fabricated, and preliminarily tested in this paper. A corresponding method has been proved effective in our previous studies. This system utilizes a light-emitting diode (LED) to excite fluorescent labels and a spectrometer to record the fluorescence signal from samples. A rotation stage for positioning and switching samples was innovatively designed for high-throughput detection, ten at most in one single run. We also developed software based on LabVIEW for data receiving, processing, and the control of the whole system. In the test of using a pure quantum dot (QD) solution as a standard sample, detection results from this home-made system were highly-relevant with that from a well-commercialized product and even slightly better reproducibility was found. And in the test of three typical kinds of food-borne pathogens, fluorescence signals recorded by this system are highly proportional to the variation of the sample concentration, with a satisfied limit of detection (LOD) (nearly 102–103 CFU·mL−1 in food samples). Additionally, this instrument system is low-cost and easy-to-use, showing a promising potential for on-site rapid detection of food-borne pathogens.

Pulsed electric field (PEF) technology is a promising nonthermal processing techniques that can be utilized to inactivate microorganisms in liquid food with high-voltage PEF. Herein, a high-voltage solid-state switch consisting of 64 insulated gate bipolar transistors (IGBTs) connected in series was designed and developed for the PEF treatment. Regarding the unbalanced sharing of voltage in series-connected IGBTs, the resistor-capacitor-diode snubber circuit was specifically used and investigated in terms of model of parameters. Furthermore, using gate drivers and optic fiber, the driving circuit and protection circuit were designed and validated. A 50-kV isolation level power supply was built in order to provide 16 independent IGBT stacks with 24 V of power each. The results show that the developed switch works adequately a delay time of 380 ns with 35.8-kV voltage and 44.8-A current capacity. Moreover, the response time of the short-circuit protection is acceptable as well with a reaction time of under 7 mu s. In conclusion, the switch designed for PEF treatment of liquid food performs within set parameters and is ready for pilot-scale processing capability.

In order to reduce the unavoidable grain losses during harvesting, the combine harvester's operational parameters should be adjusted accordingly. So, it is important to develop a real-time sensor which can monitor the grain losses. A grain impact sensor utilizing crossed piezoelectric polyvinylidene fluoride (PVDF) films as sensitive material is described. This sensor is composed of two crossed layers of sensor unit arrays, a damping layer and a support plate. The two layers are insulated from each other but can detect the impact simultaneously. The sensor unit arrays of those two layers are perpendicular and the sensor units in each layer are independent and parallel. Each sensor unit has its independent signal processing circuit, which is composed of charge amplifier, band-pass filter, envelope detector and voltage comparator. Two signals from two layers presented a two-dimensional impact position information through multi-sensor fusion technology. The sensor can obtain the spatial distribution of grain loss accurately to reduce the error-recognition ratio. Moreover, the grain impact sensor was simulated by finite element method to obtain the best number and size of the sensor units for higher sensitivity, detection speed, stress transfer efficiency, deformation transfer efficiency.