A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid(PSTVd) in tomato in China

Several viroids in the genus Pospiviroid can infect tomato(Solanum lycopersicum)and cause severe diseases,posing a serious threat to tomato production.For simultaneous detection of six tomato-infecting pospiviroids-columnea latent viroid(CLVd),pepper chat fruit viroid(PCFVd),potato spindle tuber viroid(PSTVd),tomato apical stunt viroid(TASVd),tomato chlorotic dwarf viroid(TCDVd),and tomato planta macho viroid(TPMVd),we developed a universal probe based on a highly conserved 61 nt long sequence shared among them.Compared with their specific probes,the universal probe has a similar,though slightly reduced,detection sensitivity and has the advantages of simple and cost-effective preparation and simultaneous detection of the six pospiviroids.In addition,the universal probe was used in dot-blot hybridization assays for a large-scale survey of viroid(s)in tomato plantings in China.Only PSTVd was detected in a few greenhouse-planted tomato plants.Sequence analysis revealed that these tomato PSTVd isolates may have been introduced from tomato seeds imported from abroad.

Simultaneous detection of CH_(4)and CO_(2)through dual modulation off-axis integrated cavity output spectroscopy

This study established a novel method for the simultaneous detection of two-component gases.Radio frequency(RF)white noise disturbance laser current and wavelength modulation were simultaneously used to improve the off-axis integrated cavity output spectroscopy technique,and a high-precision dual modulation OA-ICOS(RF-WM-OA-ICOS)system was established.The two laser beams were coupled into one laser beam that was applied incident to the cavity of RF-WM-OA-ICOS system.The second harmonic signals of CH_(4)and CO_(2)gas simultaneously appeared in the rising or falling edge of a triangular wave.This method was used to measure CH_(4)and CO_(2)with different concentrations.The results indicated that the proposed system has high stability and can accurately and simultaneously measure the concentrations of CH_(4)and CO_(2),with an optimal integration time of 220 s.The minimum detection limit was 10 ppb for CH_(4)and 1.5 ppm for CO_(2).The corresponding noise equivalent absorption sensitivity values were calculated as 2.67×10^(-13)cm^(-1)·Hz^(-1/2)and 5.18×10^(-11)cm^(-1)·Hz^(-1/2),respectively.The proposed dual-component gas simultaneous detection method can also be used for high-precision simultaneous detection of other gases.Therefore,this study may serve as a reference for developing portable multicomponent gas analyzers.

Simultaneous detection of different serum pepsinogens and its primary application

AIM: To develop the simple, rapid and sensitive dual-label time-resolved fluoroimmunoassay for pepsinogens in human serum.METHODS: Based on two-site sandwich protocol, mono-clonal antibodies (McAbs) against pepsinogen Ⅰ (PG Ⅰ) and PG Ⅱ were co-coated in 96 microtitration wells, and tracer McAbs against PG Ⅰ and PG Ⅱ were labeled with europium (Eu) and samarium (Sm) chelate, respectively. Diluted serum samples of Eu3+- and Sm3+-McAbs were added into microtitration wells simultaneously. After washing, fluorescence of bound Sm3+ and Eu3+ tracers was detected. RESULTS: The detection limit was 0.2 μg/L for PG Ⅰ and 0.05 μg/L for PG Ⅱ. The assay range was 5.0-320.0 μg/Lfor PG Ⅰ and 1.0-55.0 μg/L for PG Ⅱ. The average re-covery rate was 102.7% for PG Ⅰ and 98.8% for PG Ⅱ. Sera from healthy controls and patients with gastric dis-ease were analyzed. The PG detected by dual-label as-say was in good agreement with that detected by single-label assay or by enzyme-linked immunosorbent assay. CONCLUSION: Dual-label assay can provide high-throughput serological screening for gastric diseases.

Establishment of Multiplex PCR for Simultaneous Detection of Four Venereal Pathogens

Venereal diseases are considered to be the most prevalent infectious diseases in the worldwide. China is now faced with a year-by-year increasing incidence of sexually transmitted diseases （STD）, which are spreading from high-risk groups to the general population. Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum and herpes simplex virus-2 （HSV-2） are always regarded as the most common venereal pathogens. The ＂golden standard＂ for testing Neisseria gonorrhoeae remains to be bacteria culture or microscopic examination.

Gold Nanoparticles/Thermochromic Composite Film on Screen-Printed Electrodes for Simultaneous Detection of Protein and Temperature

In this study, gold nanoparticles and thermochromic composite films modified screen-printed carbon electrodes (TM-AuNPsSPCEs) were developed as a platform for the simultaneous detection of protein and temperature. The TM-AuNPs composited film had better sensitivity resulting from the gold nanoparticles amplification effect. A phase transition model analysis of TM-AuNPs films found that the TM-AuNPs films had three-phase transition intervals (<45℃, 45℃ to 80℃ and >80℃) which accommodated the temperature requirements for protein denaturation. When used to detect different concentrations of haemoglobin (Hb) solution, the TM-AuNPs modified SPCEs had a better sensitivity in detecting the different concentrations in comparison to TM and AuNP modified SPCEs which showed no clear sensitivity towards the different Hb concentrations. The dual detection and excellent sensitivity show a good application prospect for the study of the TM-AuNPs composite film.

An integrated microfluidic device for the simultaneous detection of multiple antibiotics

Residual antibiotics in food pose a serious long-term threat to human health.Therefore,an on-site visualization method for antibiotic detection is required.However,the requirements of traditional antibiotic testing methods in terms of operator proficiency and equipment cost hinder the rapid point-of-caretesting detection of suspected samples.Herein,we reported an integrated microfluidic device combining a microfluidic chip containing cruciform valves with immunochromatographic strips for the rapid detection of multiple antibiotics in milk.The rapid qualitative and quantitative analysis of four types of antibiotics(sulfonamides,β-lactams,streptomycin,and tetracyclines)was performed using mobile phone photography and mobile phone application analysis.The detection time was maintained at 10 min.The limits of detection(LODs)for the four antibiotics were 0.15,0.12,0.25,and 0.29 ng/mL,respectively,and the selectivity for the different antibiotics was observed even in a highly complex matrix.This device successfully integrated separation and real-time detection onto a chip and might provide a promising perspective for the detection of multiple antibiotics in milk.

Simultaneous detection of enteroviruses from surface waters by real-time RT-PCR with universal primers

In order to realize simultaneous quantitative detection of various enteroviruses from water samples, a real-time reverse transcriptionpolymerase chain reaction （real-time RT-PCR） method was developed with universal primer pairs designed based on the highly conserved non-coding region sequences of genome targeting poliovirus, coxsackievirus and enterovirus 71. The recombinant plasmid was constructed as enterovirus DNA standard by cloning poliovirus cDNA into a pMD18-T vector. The real-time RT-PCR method utilizing SYBR Green I was optimized. As a result of a series of examinations, the detection limit of the method was found to be 2.31 genome equivalent copy （GEC）/μL, the intraand inter-assay variations were lower than 2% and 5%, respectively, and enteroviruses were well distinguished from other microorganisms. There was a good linear relationship （r 2 = 0.997） between the logarithm of viral density and cycle threshold in a wide range of 2.31 × 10 0 to 2.31 × 10 9 GEC/μL. The validity of the method was further proved by its application for the detection of enteroviruses from various practical water samples.

Structure regulation for ultra-high luminescence quantum yield lanthanide complex and simultaneous detection of cancer marker and ferrous ion

The exploitation of a highly selective and sensitive probe to detect both cancer marker and metal ion is of great importance.In this work,the "one stone two bird" agent of 1,10-phenanthroline(phen) is designed to disrupt the polymeric lanthanide MOFs(LnMOFs,[Ln(CHO_(2))_(3)]n,Ln=Tb,la;Eu,1 b,CHO_(2)=formic acid) {[Ln(CHO_(2))_(4)·(C_(2) H_(8) N)]n,Ln=Y,2 a;Gd,2 b;Dy,2 c,C_(2)H_(8) N=dimethylamine}) into a soluble mononuclear species [Ln(phen)_(2)(NO_(3))_(3),Ln=Tb,3 a;Eu,3 b] as well as to provide an antenna for efficient photons absorption,resulting in an ultra-high luminescence quantum yield(QY,90%) europium complex.The luminescence QY is among the highest record of monomeric(zero-dimensional) lanthanide complexes.Furthermore,mononuclear Tb3+complex(3 a) functions as a multiplex sensor towards both Fe^(2+)and cancer marker of 5-hydroxyindole-3-acetic acid(5-HIAA).Importantly,the limit of detection(LOD)for sensing 5-HIAA is an ultra-sensitive value of 1 × 10 s mol/L,which is even lower than that necessary for the early diagnosis of carcinoid tumors.More interestingly,sensing results in simulated urine reveals that 3 a has potential application for early diagnosis in the clinic.

Simultaneous Multi-vehicle Detection and Tracking Framework with Pavement Constraints Based on Machine Learning and Particle Filter Algorithm

Due to the large variations of environment with ever-changing background and vehicles with different shapes, colors and appearances, to implement a real-time on-board vehicle recognition system with high adaptability, efficiency and robustness in complicated environments, remains challenging. This paper introduces a simultaneous detection and tracking framework for robust on-board vehicle recognition based on monocular vision technology. The framework utilizes a novel layered machine learning and particle filter to build a multi-vehicle detection and tracking system. In the vehicle detection stage, a layered machine learning method is presented, which combines coarse-search and fine-search to obtain the target using the AdaBoost-based training algorithm. The pavement segmentation method based on characteristic similarity is proposed to estimate the most likely pavement area. Efficiency and accuracy are enhanced by restricting vehicle detection within the downsized area of pavement. In vehicle tracking stage, a multi-objective tracking algorithm based on target state management and particle filter is proposed. The proposed system is evaluated by roadway video captured in a variety of traffics, illumination, and weather conditions. The evaluating results show that, under conditions of proper illumination and clear vehicle appearance, the proposed system achieves 91.2% detection rate and 2.6% false detection rate. Experiments compared to typical algorithms show that, the presented algorithm reduces the false detection rate nearly by half at the cost of decreasing 2.7%–8.6% detection rate. This paper proposes a multi-vehicle detection and tracking system, which is promising for implementation in an on-board vehicle recognition system with high precision, strong robustness and low computational cost.

Fluorescence Nanobiosensor for Simultaneous Detection of Multiple Veterinary Drugs in Chicken Samples

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.

An Approach to the Simultaneous Detection of Multiple Biomarkers for the Early Diagnosis of Liver Cancer Using Quantum Dot Nanoprobes

Biomarker-based early diagnosis of liver cancer is of high clinical value for reducing the mortality rate.However,it has been challenging to establish early detection methods with a single biomarker such as alpha-fetoprotein(AFP)because of limited diagnostic sensitivity and specificity.Therefore,developing multiplexed biomarker detection assays is crucially important for early diagnosis.Yet,simultaneous detection methods involving three or more biomarkers have been scarce.Here we suggest employing the serological biomarker panel of glypican-3(GPC3),dickkopf-1(DKK1),and AFP for liver cancer detection.We present a rapid simultaneous detection approach for the biomarker panel labeled with three fluorescent quantum dot nanoprobes(emission wavelengths at 565 nm,605 nm,and 655 nm).As a proof-of-concept,simultaneous fluorescence detection of the biomarker panel was demonstrated using mixed reference samples containing human recombinant GPC3,DKK1,and AFP antigens.Our simultaneous detection approach conferred a linear range of 0.625–2.5 ng·mL^(-1)for the entire biomarker panel,which merits further clinical validation for the simultaneous and accurate determination of the biomarker panel in human serum samples.

Simultaneous electrochemical DNA hybridization assay for PAT and FMV 35S gene sequence using quantum dots as labels

An electrochemical method for the simultaneous detection of two different DNA sequences from PAT and FMV 35S gene sequence using CdS and PbS quantum dots （QDs） as labels was described. The QDs were readily functionalized with oligonucleotides as electrochemical DNA probes and selectively hybridized to the complementary sequences immobilized on the microplate. The QDs anchored on the hybrids were dissolved in the solution by the oxidation of HNO3 and further detected by a sensitive differential pulse anodic stripping voltammetric method （DPASV）. The DPASV signals of the oxidation of Cd^2＋ and Pb^2＋ ions present in the solution were different and reflected the identity of corresponding ssDNA targets sequences.

Detection of seven phytohormones in peanut tissues by ultra-highperformance liquid chromatography-triple quadrupole tandem mass spectrometry

Development of highly sensitive and reliable method for detection of phytohormones is of great significance to study plant hormones and agricultural production.In this study,an ultra-high-performance liquid chromatography-mass spectrometry/mass spectrometry method was established for separation and quantification of trans-zeatin,trans-zeatin riboside,gibberellin A3,indol-3-acetic acid,salicylic acid,abscisic acid,and jasmonic acid(JA) without any label.The sepa ration was performed on an Agilent Explus Plus C18 column by using methanol and water as mobile phases with gradient elution.The target compounds were confirmed and quantified by mass spectrum via positive electrospray ionization for trans-zeatin,transzeatin riboside,indole-3-acetic acid,and via negative electrospray ionization for gibberellin3,salicylic acid,abscisic acid,and JA.The limits of detection ranged from 0.0127 ng L^-1 for gibberellin A3(GA3) to 33.26 ng L^-1 for JA and were lower than the currently reported values in literature.The proposed method was applied for qualitative and quantitative analyses of phytohormones in peanut gynophores and pods.The recoveries of the spiked phytohormones ranged from 80.20 to102.56%.The contents of seven endogenous hormones varied specifically in different development stages of peanuts.This study provides a highly sensitive and selective detection method for hormones and elucidates the growth and development of the gynophore and peanut fruit,which are controlled by seven endogenous hormones.

An immunoassay based on lab-on-a-chip for simultaneous and sensitive detection of clenbuterol and ractopamine

Both clenbuterol(CLB)and ractopamine(RAC)areβ-adrenergic agonists.After long-term excessive intake,there will be adverse reactions such as headache,chest tightness,limb numbness,and serious lifethreatening.Simultaneous detection of CLB and RAC in related samples is of great importance for human health.In this work,we outline a microfluidics-based indirect competitive immunoassay(MICI)system that can sensitively detect residual CLB and RAC in pork,swine blood and swine urine.The rapid detection of multiple samples can be achieved in one chip,which greatly improves the detection efficiency.This method has good stability and reproducibility and the microfluidic chips are easy to manufacture.The linear ranges for CLB and RAC detection by MICI are 0.1-2.5 ng/mL and 0.1-5 ng/mL,and the limits of detection(LODs)are 0.094 ng/mL and 0.091 ng/mL,respectively.This straightforward and portable immunoassay system provides a good platform for rapid detection of harmful substances in food samples.

Simultaneous Fluorescence Detection of Mercury(Ⅱ) and Silver Ions Based on Rhodamine B Isothiocyanate and 5-Carboxyfluorescein-ss DNA Modified Probe

A new fluorescence sensor is developed for simultaneous detection of Hg^2＋ and Ag^＋. During the detection process,Hg^2＋ forms complexes with the fluorescent dye rhodamine B isothiocyanate（RBITC） modified onto the surface of gold nanoparticles（Au NPs）,resulting in RBITC＇s displacement from the surface of Au NPs and the recovery of fluorescence. Meanwhile,Ag^＋ forms ＂C-Ag^＋-C＂ complex with C-rich 5-carboxyfluorescein（FAM）-ss DNA modified onto the surface of Au NPs,which keeps the fluorescent dye FAM close to the Au NPs and results in quench of fluorescence. The experimental results show a wide linear range and a good sensitivity. The limit of detection is 1.06 nmol/L for Ag^＋ and 0.48 nmol/L for Hg^2＋. This detection method is not only easy to operate but also efficient.

Advances in Multiplexed Microfluidics for Infectious Disease Detection

Microfluidics enables miniaturization,functionality,high throughput and reproducibility of multipathogen detection.Multiplexed microfluidic devices are electrochemical sensor–based,optical sensor–based,immunosensor-based and paper-based multiplexed microfluidics.However,the simultaneous detection ofmultiple pathogens is limited because of the complexity and diversity of infectious disease sources and mutual interference among analytes.This review provides an overview of recent advances in developing multiplex diagnostic microfluidic devices for detecting infectious diseases and discusses practical issues and perspectives.This review also coversmicrofluidic nucleic acid amplification strategies to improve detection sensitivity.Finally,we discuss the limitations and challenges in the design of multiplexed microfluidics.

OFDR Based Distributed Temperature Sensor Using the Three-Channel Simultaneous Radio-Frequency Lock-ln Technique

Weak signal detection for single-mode fiber-optic distributed temperature sensor （DTS） is a key technology to achieve better performance. A hybrid technique combining the incoherent optical frequency domain reflectometry （IOFDR） and the three-channel simultaneous radio-frequency （RF） lock-in amplifier （LIA） is presented to improve the signal-to-noise ratio （SNR） of the measured spontaneous Raman backscattered light. The field programmable gate array （FPGA） based RF-LIA is designed with a novel and simple structure. The measurement frequency range is achieved from 1 kHz to 100 MHz. Experimental results show that the backscattered light signal of picowatt level can be detected with high SNR. With a 2.5kin single-mode fiber, a 1064nm laser source, and the measurement time of 500 s, this sensing system can reach a spatial resolution of 0.93 m and a temperature resolution of about 0.2℃.

Simultaneous and Spatial Quantification of Telomerase Activity and DNA Methylation in Living Cells by a Deformable Satellite Nanocapsule

Telomerase plays an essential role in many biological processes.DNA methylation regulates the expression of many genes,including telomerase.Here,we propose a deformable satellite nanocapsule fluorescein isothiocyanate(FITC)-hollowbowl mesoporous organicsilica@gold nanoparticles-methyl-CpG-binding protein 2(MECP 2)-silver nanoclusters(FHBMO@AMA),for simultaneous quantitative detection of both cytoplasmic telomerase activity and the degree of DNA methylation.This strategy enabled spatial-based detection in cells.The total cytoplasmic telomerase activity was detected by fluorescence energy resonance transfer(FRET)between FHBMO and gold nanoparticles(Au NPs),while the DNA methylation in the nucleus was detected by enhanced fluorescence of silver nanoclusters(Ag NCs).Furthermore,FHBMO@AMA could intuitively distinguish between the differences in telomerase expression in cells during the DNA synthesis period at the mitotic phase(S/M)of the cell cycle.Interestingly,the ratio of the two detections(telomerase activity/DNA methylation)significantly correlated with the efficacy of anticancer drugs.At the same time,there was no apparent linear relationship between any single detection target and the efficacy of the anticancer drugs.Therefore,based on the relationship between telomerase activity and DNA methylation,our newly developed approach serves as new and feasible method for evaluating the efficacy of anticancer drugs,thereby,extending the technology toolbox for precision in medical and pharmaceutical analysis of drug potency.

（4-Ferrocenylethyne） phenylamine on Graphene as the Signal Amplificator to Determinate Dopamine and Acetaminophen Simultaneously

A novel （4-ferrocenylethyne） phenylamine functionalized graphene sheets （FEPA-GR）, coupling with chitosan （CS） were used as a signal amplification platform for simultaneous and sensitive determination of dopamine （DA） and acetaminophen （AC）. In this work, FEPA used as electron transfer mediator can be immobilized on GR surface via strong π-π stacking interaction between the conjugate chain of FEPA and GR, which effectively prevents FEPA electron mediator leaking from the electrode surface and amplified the signal. Transmission electron microscopy, FT-IR spectroscopy, UV-vis spectroscopy and electrochemical experiments results are all demonstrated the strong π-π stacking interaction between FEPA and GR. The resulted biosensor exhibited a fast response, remarkable electrocatalytic activity, perfect anti-interference ability and good stability for simultaneous detection of DA and AC. Under the optimum conditions, the oxidation peak currents of DA and AC were linearly correlated to their concentrations in the range of 2.0-135.0 μmol·L-1 and 0.3--80.0μmol·L 1, respectively. The lower detection limits for DA and AC were 0.30 and 0.05 μmol·L-1, respectively. The feasibility of the proposed method was validated by successfully applied to the simultaneous determination of DA and AC in serum samples with the standard addition method.