The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, method...The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout (KO) cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.展开更多
Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for l...Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.展开更多
Background Recently, due to the rapid development of proteomic techniques, great advance has been made in many scientific fields. We aimed to use magnetic beads (liquid chip) based matrix-assisted laser desorption/i...Background Recently, due to the rapid development of proteomic techniques, great advance has been made in many scientific fields. We aimed to use magnetic beads (liquid chip) based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology to screen distinctive biomarkers for lung adenocarcinoma (adCA), and to establish the diagnostic protein profiles. Methods Using weak cation exchange magnetic beads (MB-WCX) to isolate and purify low molecular weight proteins from sera of 35 lung adCA, 46 benign lung diseases (BLDs) and 44 healthy individuals. The resulting spectra gained by anchor chip-MALDI-TOF-MS were analyzed by ClinProTools and a pattern recognition genetic algorithm (GA). Results In the working mass range of 800-10 000 Da, 99 distinctive peaks were resolved in lung adCA versus BLDs, while 101 peaks were resolved in lung adCA versus healthy persons. The profile gained by GA that could distinguish adCA from BLDs was comprised of 4053.88, 4209.57 and 3883.33 Da with sensitivity of 80%, specificity of 93%, while that could separate adCA from healthy control was comprised of 2951.83 Da and 4209.73 Da with sensitivity of 94%, specificity of 95%. The sensitivity provided by carcinoembryonic antigen (CEA) in this experiment was significantly lower than our discriminatory profiles (P 〈0.005). We further identified a eukaryotic peptide chain release factor GTP-binding subunit (eRF3b) (4209 Da) and a complement C3f (1865 Da) that may serve as candidate biomarkers for lung adCA. Conclusion Magnetic beads based MALDI-TOF-MS technology can rapidly and effectively screen distinctive proteins/polypeptides from sera of lung adCA patients and controls, which has potential value for establishing a new diagnostic method for lung adCA.展开更多
Aflatoxin B_(1)(AFB_(1))is one of the most toxic,mutagenic and carcinogenic mycotoxin,widely exists in contaminated food,grains and feedstuff products.In this study,a novel magnetic beads multicolor colorimetric immun...Aflatoxin B_(1)(AFB_(1))is one of the most toxic,mutagenic and carcinogenic mycotoxin,widely exists in contaminated food,grains and feedstuff products.In this study,a novel magnetic beads multicolor colorimetric immunoassay(MBMCIA)based on Au@Ag nanorods(Au@Ag NRs)is proposed to visual detect ultralow concentration of AFB_(1)with high-resolution by the naked-eye.To design the MBMCIA system,AFB_(1)-BSA conjugates were first coated on the surface of magnetic beads(MBs),then alkaline phosphatase(ALP)as a bridge between immunoassay a nd color reaction was used for catalytic hydrolysis of ascorbic acid-phosphate to generate reductive ascorbic acid.Finally,the yielded ascorbic acid could reduce silver ions to grow a silver coating on the surface of gold nanorods to generate Au@Ag NRs,which leads to the bule-shifted longitudinal absorption peak of Au NRs,accompanying with a series of perceptible color change.Under the optimal conditions,the proposed MBMCIA exhibited go od sensitivity and specificity for the detection of AFB_(1)with the detection limit as low as 5.7 pg/mL Meanwhile,the MBMCIA was also applied for the analysis of AFB_(1)in spiked wheat samples,the obtained recoveries range from 99.1%to 104.3%with relative standard deviation(RSD)less than 7.05%were acceptable.The proposed MBMCIA integrates separated,enriched,anti-interfe rence and signal read-out into one,which opens up a new avenue for an on-site visual food safety inspection or environmental monitoring.展开更多
Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membra...Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.展开更多
A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads wit...A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA A PCR amplification system and a small HLA A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe 2O 3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab on a chip.展开更多
Heavy metal ion is one of the major environmental pollutants.In this study,a Cu(Ⅱ)ions imprinted magnetic chitosan beads are prepared to use chitosan as functional monomer,Cu(Ⅱ)ions as template,Fe_(3)O_(4) as magnet...Heavy metal ion is one of the major environmental pollutants.In this study,a Cu(Ⅱ)ions imprinted magnetic chitosan beads are prepared to use chitosan as functional monomer,Cu(Ⅱ)ions as template,Fe_(3)O_(4) as magnetic core and epichlorohydrin and glutaraldehyde as crosslinker,which can be used for removal Cu(Ⅱ)ions from wastewater.The kinetic study shows that the adsorption process follows the pseudosecond-order kinetic equations.The adsorption isotherm study shows that the Langmuir isotherm equation best fits for the monolayer adsorption processes.The selective adsorption properties are performed in Cu(Ⅱ)/Zn(Ⅱ),Cu(Ⅱ)/Ni(Ⅱ),and Cu(Ⅱ)/Co(Ⅱ)binary systems.The results shows that the ⅡMCD has a high selectivity for Cu(Ⅱ)ions in binary systems.The mechanism of ⅡMCD recognition Cu(Ⅱ)ions is also discussed.The results show that the ⅡMCD adsorption Cu(Ⅱ)ions is an enthalpy controlled process.The absolute value of DH(Cu(Ⅱ))and DS(Cu(Ⅱ))is greater than DH(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ))and DS(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ)),respectively,this indicates that the Cu(Ⅱ)ions have a good spatial matching with imprinted holes on ⅡMCD.The FTIR and XPS also demonstrates the strongly combination of function groups on imprinted holes in the suitable space position.Finally,the ⅡMCD can be regenerated and reused for 10 times without a significantly decreasing in adsorption capacity.This information can be used for further application in the selective removal of Cu(Ⅱ)ions from industrial wastewater.展开更多
To investigate the technique of sorting high-purity precartilaginous stem cells from rat's perichondrium, neonatal rat's perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast grow...To investigate the technique of sorting high-purity precartilaginous stem cells from rat's perichondrium, neonatal rat's perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast growth factor receptor-3 (anti-FGFR-3), and the labeled cells were separated from the suspension in the magnetic field by immuno-beads coated with the second antibody, Purity of the sorted neural stem cells was found to be 93.0 %-99.0 %, with living cells amounting to 80 % -85 %. The magnetic cell sorting system could effectively separate precartilaginous stem cells from perichondrium cell suspensions.展开更多
In this study, a computer code is developed to numerically investigate a magnetic bead micromixer under different conditions. The micromixer consists of a microchannel and numerous micro magnetic particles which enter...In this study, a computer code is developed to numerically investigate a magnetic bead micromixer under different conditions. The micromixer consists of a microchannel and numerous micro magnetic particles which enter the micromixer by fluid flows and are actuated by an alternating magnetic field normal to the main flow. An important feature of micromixer which is not considered before by researchers is the particle entrance arrangement into the micromixer. This parameter could effectively affect the micromixer efficiency. There are two general micro magnetic particle entrance arrangements in magnetic bead micromixers: determined position entrance and random position entrance. In the case of determined position entrances, micro magnetic particles enter the micromixer at specific positions of entrance cross section. However, in a random position entrance,particles enter the microchannel with no order. In this study mixing efficiencies of identical magnetic bead micromixers which only differ in particle entrance arrangement are numerically investigated and compared.The results reported in this paper illustrate that the prepared computer code can be one of the most powerful and beneficial tools for the magnetic bead micromixer performance analysis. In addition, the results show that some features of the magnetic bead micromixer are strongly affected by the entrance arrangement of the particles.展开更多
AIM: To find new biomarkers for uveal melanoma (UM) by analyzing the serum peptidome profile. METHODS: Proteomic spectra in patients with UM before and after operation were analyzed and compared with those of hea...AIM: To find new biomarkers for uveal melanoma (UM) by analyzing the serum peptidome profile. METHODS: Proteomic spectra in patients with UM before and after operation were analyzed and compared with those of healthy controls. Magnetic affinity beads were used to capture serum peptides and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer were used to compile serum peptide profiles. RESULTS: A panel of 49 peptides were differentially expressed between UM patients and controls, of which 33 peptides were of higher intensities in patient group and 16 peptides were of higher intensities in control group. Based on combined use of these potential markers, peptides with mean molecular masses of 1467 and 9289.0 Da provide high sensitivity (83.3%), specificity (100%) and accuracy rate (93.0%) together to differentiate melanoma patients from healthy controls. At the time point of 6mo postoperatively, the levels of many peptides differentially expressed before surgery showed no more statistical difference between the patients and the control group. Fibrinogen o-chain precursors were identified as potential UM markers. CONCLUSION: We have shown that a convenient and fast proteomic technique, affinity bead separation and MALDI- TOF analysis combined with bioinformatic software, facilitates the identification of novel biomarkers for UM.展开更多
BACKGROUND Since 2017,the number of magnet ingestion cases has increased year over year in our hospital.Almost all of the ingested magnetic foreign bodies were magnetic beads,and most of the patients experienced intes...BACKGROUND Since 2017,the number of magnet ingestion cases has increased year over year in our hospital.Almost all of the ingested magnetic foreign bodies were magnetic beads,and most of the patients experienced intestinal perforations,causing substantial damage.AIM To summarize our experience with surgical treatment of multiple magnet ingestion in children.METHODS The data for general surgeries were collected from January 2010 to April 2020,and the clinical characteristics,treatment methods,and outcomes were summarized and analyzed.Several typical cases were selected and discussed.RESULTS Fifty-six cases of ingested magnetic foreign bodies were collected,of which 47 were magnetic beads.The average patient age was 4.7±3.0 years old.The number of ingested magnetic foreign bodies ranged from 2 to 73.There were 26 cases with symptoms at the time of admission,including two cases of shock.Thirteen patients were discharged successfully following conservative treatment and 43 were treated by surgery.Laparotomy was the main method of operation.Laparoscopy was used in four cases,of which three were converted to open surgery,and one was treated successfully using surgery through the navel.Postoperative complications occurred in seven cases,incision infections were observed in six,and adhesive ileus was observed in one.CONCLUSION Clinicians need to summarize their experiences with treating magnetic foreign body ingestions in detail and carry out clinical research to reduce the damage to children.展开更多
An easy method is presented to fabricate monodisperse magnetic macroporous polymer beads(MMPBs). Waterin-oil high internal phase emulsion(HIPE) is prepared by emulsifying aqueous iron ions solution in an oil phase...An easy method is presented to fabricate monodisperse magnetic macroporous polymer beads(MMPBs). Waterin-oil high internal phase emulsion(HIPE) is prepared by emulsifying aqueous iron ions solution in an oil phase containing monomers. The HIPE is introduced into a simple microfluidic device to fabricate monodisperse(water-in-oil)-in-water double emulsion droplets. The droplets serve as microreactors to synthesize Fe3O4 nanoparticles and are on-line polymerized to form MMPBs. The prepared MMPBs display uniform size, interconnected porous structure, superparamagnetic behavior and uniform distribution of Fe3O4 in polymer matrix. The MMPBs are characterized by scanning electron microscopy(SEM), Fourier transform infrared spectroscopy(FTIR), X-ray diffraction(XRD), transmission electron microscopy(TEM), vibrating sample magnetometry(VSM). We believe that this method is a universal technique in preparing macroporous nanocomposite beads.展开更多
Diarrhea,as a global public health problem,causes a large number of infections and deaths every year.Although Escherichia coli(E.coli)is one of the normal flo ra microorganisms in the human intestinal tract,it has fiv...Diarrhea,as a global public health problem,causes a large number of infections and deaths every year.Although Escherichia coli(E.coli)is one of the normal flo ra microorganisms in the human intestinal tract,it has five pathogenic bacteria types that can cause human diarrhea,known as diarrheagenic E.coli.When people are infected,rapid and accurate diagnosis,along with timely treatment,are especially important.Here,we introduce a new method to identify and analyze a large number of pathogenic strains in E.coli by multiplex PCR and barcoded magnetic bead hybridization.Results show that the detection sensitivities of enterohemorrhagic E.coli,enterotoxigenic E.coli,enteropathogenic E.coli,enteroinvasive E.coli and enteroaggregative E.coli were 1.3×10^3 CFU/mL,2×10^4 CFU/mL,4×10^4 CFU/mL,7.2×10^4 CFU/mL and 1.7 CFU/mL respectively.This method has strong specificity and high sensitivity and detects multiple target sequences in one experiment.Compared with other methods,BMB array has great application potential.展开更多
Enzyme-linked immunosorbent assay(ELISA)provides a convenient way for the detection of viral pathogens.However,conventional ELISA performed on mirowell plates suffers from poor sensitivity,laborious coating and compli...Enzyme-linked immunosorbent assay(ELISA)provides a convenient way for the detection of viral pathogens.However,conventional ELISA performed on mirowell plates suffers from poor sensitivity,laborious coating and complicated blocking procedures.Herein,we designed a sensitive colorimetric immunoassay by taking advantages of the enrichment and isolation ability of magnetic beads(MBs)and the high loading capacity of gold nanoparticles(AuNPs)for detecting respiratory syncytial virus(RSV)as a pathogen model.RSV was selectively captured and preconcentrated from samples with antibodies functionalized MBs,followed by binding with antibodies labeled AuNPs,which carrying a large amount of alkaline phosphatase(ALP)molecules for colorimetric signal amplification by catalyzing the dephosphorylation of non-colored pNPP to generate colored product pNP.After optimizing the experimental conditions based on the principle of low nonspecific signal,low cost,and high sensitivity,the analytical sensitivity of the developed immunoassay can be improved to 0.27 pg/mL,which is over sevenfold higher than that of commercially available RSV ELISA kits(2 pg/mL).In addition,the total assay time was less than 2.5 h without any pretreatment,which is much more rapid than other reported assays.Therefore,the proposed immunoassay holds great promise for the fabrication of rapid,sensitive,and economic method for the viral pathogen detection.展开更多
Neuraminidase inhibitors(NAIs)are the mainstay antiviral drugs against influenza infection.In this study,a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads(NA-MB).Aft...Neuraminidase inhibitors(NAIs)are the mainstay antiviral drugs against influenza infection.In this study,a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads(NA-MB).After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors,the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr.Twenty-four NA binding compounds were identified from the normal butanol(n-BuOH)extract of D.indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS)assisted with Compound Structure Identification(CSI):FingerID,including 12 ellagitannins,4 brevifolin derivatives,3 ellagic acid derivatives,and 4 flavonoids.Among them,9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens,and from oseltamivir sensitive and resistant influenza A virus strains.The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA,with half maximal inhibitory concentration(IC50)values of 197.9 and 125.4μmol/L,respectively.Moreover,B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin-Darby canine kidney(MDCK)cells at the concentrations of 40 and 200μmol/L.展开更多
Salmonella Enteritidis is a major public health threat of global proportions,while it is still a serious challenge to develop point-of-care detection assay.In this study,nanozymes(Fe-MOF nanoparticles)were successfull...Salmonella Enteritidis is a major public health threat of global proportions,while it is still a serious challenge to develop point-of-care detection assay.In this study,nanozymes(Fe-MOF nanoparticles)were successfully synthesized and applied as signal in a colorimetric immunoassay for naked-eye detection of Salmonella Enteritidis.After optimization,the proposed assay was able to detect Salmonella Enteritidis with a detection limit of 34 CFU/mL.The coefficients of variation(CV)of the test were less than 7.0%after 30 days storage at 4°C.The estimated recoveries in milk samples of the colorimetric immu-noassay range from 94.68 to 124%,which indicated the developed method is capable of detecting Salmonella Enteritidis in real samples.This method provides a potential platform for Salmonella detection with naked eyes,which has a significant application value for foodborne pathogen analysis at the point-of-care.展开更多
The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors. Herein, we have described a multiple genotyping method based on magnetic enrichment- multiplex PCR (ME...The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors. Herein, we have described a multiple genotyping method based on magnetic enrichment- multiplex PCR (MEM-PCR) and microarray technology. Monodisperse magnetic beads were fabricated and modified with streptavidin. Four loci on two genes (M235T and A-6G loci on AGT gene, A1298C and C677T loci on MTHFR gene) were selected to study single nucleotide polymorphisms (SNP). Target sequences of these SNP loci were amplified using Cy3-1abeled primers through multiplex PCR in one tube after the templates were enriched and purified by functional magnetic beads (MB). Four pairs of NH2- labeled probes, corresponding to each locus, were fixed on CHO-modified glass slide by covalent binding. Hybridization between target sequences and probes was performed under suitable conditions. The spotting locations on microarray and the ratio of fluorescence intensity, produced by different loci, were used to distinguish the SNP genotypes. Finally, three of gastric cancer samples were collected and genotvping analysis for these four SNP loci was carried out successfully simultaneously by this method.展开更多
Background In recent years the proportion of lung adenocarcinoma (adCA) which occurs in lung cancer patients has increased. Using laser capture microdissection (LCM) combined with liquid chip-mass spectrometry tec...Background In recent years the proportion of lung adenocarcinoma (adCA) which occurs in lung cancer patients has increased. Using laser capture microdissection (LCM) combined with liquid chip-mass spectrometry technology, we aimed to screen lung cancer biomarkers by studying the proteins in the tissues of adCA. Methods We used LCM and magnetic bead based weak cation exchange (MB-WCX) to separate and purify the homogeneous adCA cells and normal cells from six cases of fresh adCA and matched normal lung tissues. The proteins were analyzed and identified by matrix assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-OF-MS). We screened for the best pattern using a radial basic function neural network algorithm. Results About 2.895x10s and 1.584x10s cells were satisfactorily obtained by LCM from six cases of fresh lung adCA and matched normal lung tissues, respectively. The homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. Comparing the differentially expressed proteins between the lung adCA and the matched normal lung group, 221 and 239 protein peaks, respectively, were found in the mass-to-charge ration (M/Z) between 800 Da and 10 000 Da. According to ttest, the expression of two protein peaks at 7521.5 M/Zand 5079.3 M/Z had the largest difference between tissues. They were more weakly expressed in the lung adCA compared to the matched normal group. The two protein peaks could accurately separate the lung adCA from the matched normal lung group by the sample distribution chart. A discriminatory pattern which can separate the lung adCA from the matched normal lung tissue consisting of three proteins at 3358.1 M/Z, 5079.3 M/Z and 7521.5 M/Z was established by a radial basic function neural network algorithm with a sensitivity of 100% and a specificity of 100%. Conclusions Differential proteins in lung adCA were screened using LCM combined with liquid chip-mass spectrometry technology, and a biomarker model was established. It is possible that this technology is going to become a powerful tool in screening and early diagnosis of lung adCA.展开更多
The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-...The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.展开更多
基金supported by the National Basic Research Program of China(973 Program)(2015CB554103 and 2011CBA01004)
文摘The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cel nuclear transfer (SCNT) method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout (KO) cels was established, and corresponding geneticaly modiifed pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine α-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.
基金supported by the Zhejiang Foundation Public Welfare Research Project(Authorization No.:LGF19B060006)。
文摘Due to the complexity of bioactive ingredients in biological samples,the screening of target proteins is a complex process.Herein,a feasible strategy for directing protein immobilization on silica magnetic beads for ligand fishing based on SpyTag/SpyCatcher(ST/SC)-mediated anchoring is presented.Carboxyl functional groups on the surface of silica-coated magnetic beads(SMBs)were coupled with SC using the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide method,named SC-SMBs.The green fluorescent protein(GFP),as the capturing protein model,was ST-labeled and anchored at a specific orientation onto the surface of SC-SMBs directly from relevant cell lysates via ST/SC self-ligation.The characteristics of the SC-SMBs were studied via electron microscopy,energy dispersive spectroscopy,and Fourier transform infrared spectroscopy.The spontaneity and site-specificity of this unique reaction were confirmed via electrophoresis and fluorescence analyses.Although the alkaline stability of ST-GFP-ligated SC-SMBs was not ideal,the formed isopeptide bond was unbreakable under acidic conditions(0.05 M glycine-HCl buffer,pH 1e6)for 2 h,under 20%ethanol solution within 7 days,and at most temperatures.We,therefore,present a simple and universal strategy for the preparation of diverse protein-functionalized SMBs for ligand fishing,prompting its usage on drug screening and target finding.
基金This work was supported by grants from the National Natural Science Foundation of China (No. 30570795) and Program for New Century Excellent Talents in University (No. NECT-06-0845) and the Program in Science and Technology of Xi'an, Shaanxi Province (No. S F08009(1)).Acknowledgement: We are grateful to HU Xiao-hui for the technical guidance.
文摘Background Recently, due to the rapid development of proteomic techniques, great advance has been made in many scientific fields. We aimed to use magnetic beads (liquid chip) based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technology to screen distinctive biomarkers for lung adenocarcinoma (adCA), and to establish the diagnostic protein profiles. Methods Using weak cation exchange magnetic beads (MB-WCX) to isolate and purify low molecular weight proteins from sera of 35 lung adCA, 46 benign lung diseases (BLDs) and 44 healthy individuals. The resulting spectra gained by anchor chip-MALDI-TOF-MS were analyzed by ClinProTools and a pattern recognition genetic algorithm (GA). Results In the working mass range of 800-10 000 Da, 99 distinctive peaks were resolved in lung adCA versus BLDs, while 101 peaks were resolved in lung adCA versus healthy persons. The profile gained by GA that could distinguish adCA from BLDs was comprised of 4053.88, 4209.57 and 3883.33 Da with sensitivity of 80%, specificity of 93%, while that could separate adCA from healthy control was comprised of 2951.83 Da and 4209.73 Da with sensitivity of 94%, specificity of 95%. The sensitivity provided by carcinoembryonic antigen (CEA) in this experiment was significantly lower than our discriminatory profiles (P 〈0.005). We further identified a eukaryotic peptide chain release factor GTP-binding subunit (eRF3b) (4209 Da) and a complement C3f (1865 Da) that may serve as candidate biomarkers for lung adCA. Conclusion Magnetic beads based MALDI-TOF-MS technology can rapidly and effectively screen distinctive proteins/polypeptides from sera of lung adCA patients and controls, which has potential value for establishing a new diagnostic method for lung adCA.
基金supported by the National Natural Science Foundation of China(Nos.21804022,21964003 and 81773894)the Natural Science Foundation of Jiangxi Province(No.20202BABL213019)+1 种基金the Science and Technology Project of the Education Department of Jiangxi Province of China(No.GJJ190775)the Special Graduate Student Innovation Fund of Jiangxi Province(No.CX190013)。
文摘Aflatoxin B_(1)(AFB_(1))is one of the most toxic,mutagenic and carcinogenic mycotoxin,widely exists in contaminated food,grains and feedstuff products.In this study,a novel magnetic beads multicolor colorimetric immunoassay(MBMCIA)based on Au@Ag nanorods(Au@Ag NRs)is proposed to visual detect ultralow concentration of AFB_(1)with high-resolution by the naked-eye.To design the MBMCIA system,AFB_(1)-BSA conjugates were first coated on the surface of magnetic beads(MBs),then alkaline phosphatase(ALP)as a bridge between immunoassay a nd color reaction was used for catalytic hydrolysis of ascorbic acid-phosphate to generate reductive ascorbic acid.Finally,the yielded ascorbic acid could reduce silver ions to grow a silver coating on the surface of gold nanorods to generate Au@Ag NRs,which leads to the bule-shifted longitudinal absorption peak of Au NRs,accompanying with a series of perceptible color change.Under the optimal conditions,the proposed MBMCIA exhibited go od sensitivity and specificity for the detection of AFB_(1)with the detection limit as low as 5.7 pg/mL Meanwhile,the MBMCIA was also applied for the analysis of AFB_(1)in spiked wheat samples,the obtained recoveries range from 99.1%to 104.3%with relative standard deviation(RSD)less than 7.05%were acceptable.The proposed MBMCIA integrates separated,enriched,anti-interfe rence and signal read-out into one,which opens up a new avenue for an on-site visual food safety inspection or environmental monitoring.
基金supported in part by research grants from the China Postdoctoral Science Foundation(2019M663446 to ZZ)the Postdoctoral Program of the Natural Science Foundation of Chongqing,China(cstc2019jcyj-bsh0006 to ZZ)+6 种基金WW was supported by the Medical Scientist Training Program of the National Institutes of Health(T32 GM007281)This project was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430TCH was supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund.Funding sources were not involved in the study designin the collection,analysis and interpretation of datain the writing of the reportand in the decision to submit the paper for publication.
文摘Plasmid DNA(pDNA)isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research.Almost all pDNA purification in-volves disruption of bacteria,removal of membrane lipids,proteins and genomic DNA,purifi-cation of pDNA from bulk lysate,and concentration of pDNA for downstream applications.While many liquid-phase and solid-phase pDNA purification methods are used,the final pDNA preparations are usually contaminated with varied degrees of host RNA,which cannot be completely digested by RNase A.To develop a simple,cost-effective,and yet effective method for RNA depletion,we investigated whether commercially available size selection magnetic beads(SSMBs),such as Mag-Bind®TotalPure NGS Kit(or Mag-Bind),can completely deplete bacterial RNA in pDNA preparations.In this proof-of-principle study,we demonstrated that,compared with RNase A digestion and two commercial plasmid affinity purification kits,the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits.We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples.Furthermore,the Mag-bind-based SSMB method costs only 5-10%of most commercial plasmid purification kits on a per sample basis.Thus,the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.
文摘A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA A PCR amplification system and a small HLA A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe 2O 3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab on a chip.
文摘Heavy metal ion is one of the major environmental pollutants.In this study,a Cu(Ⅱ)ions imprinted magnetic chitosan beads are prepared to use chitosan as functional monomer,Cu(Ⅱ)ions as template,Fe_(3)O_(4) as magnetic core and epichlorohydrin and glutaraldehyde as crosslinker,which can be used for removal Cu(Ⅱ)ions from wastewater.The kinetic study shows that the adsorption process follows the pseudosecond-order kinetic equations.The adsorption isotherm study shows that the Langmuir isotherm equation best fits for the monolayer adsorption processes.The selective adsorption properties are performed in Cu(Ⅱ)/Zn(Ⅱ),Cu(Ⅱ)/Ni(Ⅱ),and Cu(Ⅱ)/Co(Ⅱ)binary systems.The results shows that the ⅡMCD has a high selectivity for Cu(Ⅱ)ions in binary systems.The mechanism of ⅡMCD recognition Cu(Ⅱ)ions is also discussed.The results show that the ⅡMCD adsorption Cu(Ⅱ)ions is an enthalpy controlled process.The absolute value of DH(Cu(Ⅱ))and DS(Cu(Ⅱ))is greater than DH(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ))and DS(Zn(Ⅱ),Ni(Ⅱ),Co(Ⅱ)),respectively,this indicates that the Cu(Ⅱ)ions have a good spatial matching with imprinted holes on ⅡMCD.The FTIR and XPS also demonstrates the strongly combination of function groups on imprinted holes in the suitable space position.Finally,the ⅡMCD can be regenerated and reused for 10 times without a significantly decreasing in adsorption capacity.This information can be used for further application in the selective removal of Cu(Ⅱ)ions from industrial wastewater.
基金This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30170944).
文摘To investigate the technique of sorting high-purity precartilaginous stem cells from rat's perichondrium, neonatal rat's perichondrium cells suspensions were incubated with monoclone antibody of anti-fibroblast growth factor receptor-3 (anti-FGFR-3), and the labeled cells were separated from the suspension in the magnetic field by immuno-beads coated with the second antibody, Purity of the sorted neural stem cells was found to be 93.0 %-99.0 %, with living cells amounting to 80 % -85 %. The magnetic cell sorting system could effectively separate precartilaginous stem cells from perichondrium cell suspensions.
文摘In this study, a computer code is developed to numerically investigate a magnetic bead micromixer under different conditions. The micromixer consists of a microchannel and numerous micro magnetic particles which enter the micromixer by fluid flows and are actuated by an alternating magnetic field normal to the main flow. An important feature of micromixer which is not considered before by researchers is the particle entrance arrangement into the micromixer. This parameter could effectively affect the micromixer efficiency. There are two general micro magnetic particle entrance arrangements in magnetic bead micromixers: determined position entrance and random position entrance. In the case of determined position entrances, micro magnetic particles enter the micromixer at specific positions of entrance cross section. However, in a random position entrance,particles enter the microchannel with no order. In this study mixing efficiencies of identical magnetic bead micromixers which only differ in particle entrance arrangement are numerically investigated and compared.The results reported in this paper illustrate that the prepared computer code can be one of the most powerful and beneficial tools for the magnetic bead micromixer performance analysis. In addition, the results show that some features of the magnetic bead micromixer are strongly affected by the entrance arrangement of the particles.
基金Supported by the National Natural Science Foundation of China(No.81570891No.81272981)+2 种基金the Beijing Natural Science Foundation(No.7151003)Advanced Health Care Professionals Development Project of Beijing Municipal Health Bureau(No.2014-2-003)Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support(No.ZYLX201307)
文摘AIM: To find new biomarkers for uveal melanoma (UM) by analyzing the serum peptidome profile. METHODS: Proteomic spectra in patients with UM before and after operation were analyzed and compared with those of healthy controls. Magnetic affinity beads were used to capture serum peptides and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer were used to compile serum peptide profiles. RESULTS: A panel of 49 peptides were differentially expressed between UM patients and controls, of which 33 peptides were of higher intensities in patient group and 16 peptides were of higher intensities in control group. Based on combined use of these potential markers, peptides with mean molecular masses of 1467 and 9289.0 Da provide high sensitivity (83.3%), specificity (100%) and accuracy rate (93.0%) together to differentiate melanoma patients from healthy controls. At the time point of 6mo postoperatively, the levels of many peptides differentially expressed before surgery showed no more statistical difference between the patients and the control group. Fibrinogen o-chain precursors were identified as potential UM markers. CONCLUSION: We have shown that a convenient and fast proteomic technique, affinity bead separation and MALDI- TOF analysis combined with bioinformatic software, facilitates the identification of novel biomarkers for UM.
文摘BACKGROUND Since 2017,the number of magnet ingestion cases has increased year over year in our hospital.Almost all of the ingested magnetic foreign bodies were magnetic beads,and most of the patients experienced intestinal perforations,causing substantial damage.AIM To summarize our experience with surgical treatment of multiple magnet ingestion in children.METHODS The data for general surgeries were collected from January 2010 to April 2020,and the clinical characteristics,treatment methods,and outcomes were summarized and analyzed.Several typical cases were selected and discussed.RESULTS Fifty-six cases of ingested magnetic foreign bodies were collected,of which 47 were magnetic beads.The average patient age was 4.7±3.0 years old.The number of ingested magnetic foreign bodies ranged from 2 to 73.There were 26 cases with symptoms at the time of admission,including two cases of shock.Thirteen patients were discharged successfully following conservative treatment and 43 were treated by surgery.Laparotomy was the main method of operation.Laparoscopy was used in four cases,of which three were converted to open surgery,and one was treated successfully using surgery through the navel.Postoperative complications occurred in seven cases,incision infections were observed in six,and adhesive ileus was observed in one.CONCLUSION Clinicians need to summarize their experiences with treating magnetic foreign body ingestions in detail and carry out clinical research to reduce the damage to children.
文摘An easy method is presented to fabricate monodisperse magnetic macroporous polymer beads(MMPBs). Waterin-oil high internal phase emulsion(HIPE) is prepared by emulsifying aqueous iron ions solution in an oil phase containing monomers. The HIPE is introduced into a simple microfluidic device to fabricate monodisperse(water-in-oil)-in-water double emulsion droplets. The droplets serve as microreactors to synthesize Fe3O4 nanoparticles and are on-line polymerized to form MMPBs. The prepared MMPBs display uniform size, interconnected porous structure, superparamagnetic behavior and uniform distribution of Fe3O4 in polymer matrix. The MMPBs are characterized by scanning electron microscopy(SEM), Fourier transform infrared spectroscopy(FTIR), X-ray diffraction(XRD), transmission electron microscopy(TEM), vibrating sample magnetometry(VSM). We believe that this method is a universal technique in preparing macroporous nanocomposite beads.
基金the National Natural Science Foundation of China(Nos.61971187,61571187,61871180)Education Department Outstanding Young Project of Hunan Province(No.18B299)。
文摘Diarrhea,as a global public health problem,causes a large number of infections and deaths every year.Although Escherichia coli(E.coli)is one of the normal flo ra microorganisms in the human intestinal tract,it has five pathogenic bacteria types that can cause human diarrhea,known as diarrheagenic E.coli.When people are infected,rapid and accurate diagnosis,along with timely treatment,are especially important.Here,we introduce a new method to identify and analyze a large number of pathogenic strains in E.coli by multiplex PCR and barcoded magnetic bead hybridization.Results show that the detection sensitivities of enterohemorrhagic E.coli,enterotoxigenic E.coli,enteropathogenic E.coli,enteroinvasive E.coli and enteroaggregative E.coli were 1.3×10^3 CFU/mL,2×10^4 CFU/mL,4×10^4 CFU/mL,7.2×10^4 CFU/mL and 1.7 CFU/mL respectively.This method has strong specificity and high sensitivity and detects multiple target sequences in one experiment.Compared with other methods,BMB array has great application potential.
基金the National Natural Science Foundation of China(NSFC,No.21535006)the National Basic Research Program of China(973 Program,No.2011CB933600)the Doctoral Scientific Research Foundation(SWU116058).
文摘Enzyme-linked immunosorbent assay(ELISA)provides a convenient way for the detection of viral pathogens.However,conventional ELISA performed on mirowell plates suffers from poor sensitivity,laborious coating and complicated blocking procedures.Herein,we designed a sensitive colorimetric immunoassay by taking advantages of the enrichment and isolation ability of magnetic beads(MBs)and the high loading capacity of gold nanoparticles(AuNPs)for detecting respiratory syncytial virus(RSV)as a pathogen model.RSV was selectively captured and preconcentrated from samples with antibodies functionalized MBs,followed by binding with antibodies labeled AuNPs,which carrying a large amount of alkaline phosphatase(ALP)molecules for colorimetric signal amplification by catalyzing the dephosphorylation of non-colored pNPP to generate colored product pNP.After optimizing the experimental conditions based on the principle of low nonspecific signal,low cost,and high sensitivity,the analytical sensitivity of the developed immunoassay can be improved to 0.27 pg/mL,which is over sevenfold higher than that of commercially available RSV ELISA kits(2 pg/mL).In addition,the total assay time was less than 2.5 h without any pretreatment,which is much more rapid than other reported assays.Therefore,the proposed immunoassay holds great promise for the fabrication of rapid,sensitive,and economic method for the viral pathogen detection.
基金financially supported by National Natural Science Foundation,China(81872830,31970884,and U1801287)the International Science&Technology Cooperation Program of Guangzhou,China(201807010022)National Natural Science Foundation of Guangdong Province,China(No.2019A1515011489)
文摘Neuraminidase inhibitors(NAIs)are the mainstay antiviral drugs against influenza infection.In this study,a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads(NA-MB).After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors,the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr.Twenty-four NA binding compounds were identified from the normal butanol(n-BuOH)extract of D.indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(HPLC-Q-TOF-MS)assisted with Compound Structure Identification(CSI):FingerID,including 12 ellagitannins,4 brevifolin derivatives,3 ellagic acid derivatives,and 4 flavonoids.Among them,9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens,and from oseltamivir sensitive and resistant influenza A virus strains.The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA,with half maximal inhibitory concentration(IC50)values of 197.9 and 125.4μmol/L,respectively.Moreover,B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin-Darby canine kidney(MDCK)cells at the concentrations of 40 and 200μmol/L.
基金WSU start up fund and China National Postdoctoral Program for Innovative Talents(BX20180364).
文摘Salmonella Enteritidis is a major public health threat of global proportions,while it is still a serious challenge to develop point-of-care detection assay.In this study,nanozymes(Fe-MOF nanoparticles)were successfully synthesized and applied as signal in a colorimetric immunoassay for naked-eye detection of Salmonella Enteritidis.After optimization,the proposed assay was able to detect Salmonella Enteritidis with a detection limit of 34 CFU/mL.The coefficients of variation(CV)of the test were less than 7.0%after 30 days storage at 4°C.The estimated recoveries in milk samples of the colorimetric immu-noassay range from 94.68 to 124%,which indicated the developed method is capable of detecting Salmonella Enteritidis in real samples.This method provides a potential platform for Salmonella detection with naked eyes,which has a significant application value for foodborne pathogen analysis at the point-of-care.
基金financially supported by the National Key Program for Developing Basic Research(No.2010CB933903)the Chinese National Key Project of Science and Technology(No.2013ZX10004103-002)+2 种基金the National Youth Science Foundation of China(No.61301043)the NSFC(Nos.61271056,61471168,61201100 and 61527806)the Economical Forest Cultivation and Utilization of 2011 Collaborative Innovation Center in Hunan Province[No.(2013)448]
文摘The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors. Herein, we have described a multiple genotyping method based on magnetic enrichment- multiplex PCR (MEM-PCR) and microarray technology. Monodisperse magnetic beads were fabricated and modified with streptavidin. Four loci on two genes (M235T and A-6G loci on AGT gene, A1298C and C677T loci on MTHFR gene) were selected to study single nucleotide polymorphisms (SNP). Target sequences of these SNP loci were amplified using Cy3-1abeled primers through multiplex PCR in one tube after the templates were enriched and purified by functional magnetic beads (MB). Four pairs of NH2- labeled probes, corresponding to each locus, were fixed on CHO-modified glass slide by covalent binding. Hybridization between target sequences and probes was performed under suitable conditions. The spotting locations on microarray and the ratio of fluorescence intensity, produced by different loci, were used to distinguish the SNP genotypes. Finally, three of gastric cancer samples were collected and genotvping analysis for these four SNP loci was carried out successfully simultaneously by this method.
基金This work was supported by grants from the National Natural Science Foundation of China (No. 30570795) and Program for New Century Excellent Talents in University (No. NECT-06-0845) and the Program in Science and Technology of Xi'an, Shaanxi Province (No. SF08009(1)).
文摘Background In recent years the proportion of lung adenocarcinoma (adCA) which occurs in lung cancer patients has increased. Using laser capture microdissection (LCM) combined with liquid chip-mass spectrometry technology, we aimed to screen lung cancer biomarkers by studying the proteins in the tissues of adCA. Methods We used LCM and magnetic bead based weak cation exchange (MB-WCX) to separate and purify the homogeneous adCA cells and normal cells from six cases of fresh adCA and matched normal lung tissues. The proteins were analyzed and identified by matrix assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-OF-MS). We screened for the best pattern using a radial basic function neural network algorithm. Results About 2.895x10s and 1.584x10s cells were satisfactorily obtained by LCM from six cases of fresh lung adCA and matched normal lung tissues, respectively. The homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. Comparing the differentially expressed proteins between the lung adCA and the matched normal lung group, 221 and 239 protein peaks, respectively, were found in the mass-to-charge ration (M/Z) between 800 Da and 10 000 Da. According to ttest, the expression of two protein peaks at 7521.5 M/Zand 5079.3 M/Z had the largest difference between tissues. They were more weakly expressed in the lung adCA compared to the matched normal group. The two protein peaks could accurately separate the lung adCA from the matched normal lung group by the sample distribution chart. A discriminatory pattern which can separate the lung adCA from the matched normal lung tissue consisting of three proteins at 3358.1 M/Z, 5079.3 M/Z and 7521.5 M/Z was established by a radial basic function neural network algorithm with a sensitivity of 100% and a specificity of 100%. Conclusions Differential proteins in lung adCA were screened using LCM combined with liquid chip-mass spectrometry technology, and a biomarker model was established. It is possible that this technology is going to become a powerful tool in screening and early diagnosis of lung adCA.
基金supported by grants from the National Natural Science Foundation of China (No.21472060)Shenzhen Municipal government (Nos.JCYJ20160301153959476 and JCYJ20160324163734374)Shenzhen Reform Commission (Disciplinary Development Program for Chemical Biology)
文摘The detection of biomarkers is of great significance in the diagnosis of numerous diseases,especially cancer.Herein,we developed a sensitive and universal fluorescent aptasensor strategy based on magnetic beads,DNA G-quadruplex,and exonuclease Ⅲ(Exo Ⅲ).In the presence of a target protein,a label-free single strand DNA(ssDNA)hybridized with the aptamer was released as a trigger DNA due to specific recognition between the aptamer and target.Subsequently,ssDNA initiates the ExoⅢ-aided recycling to amplify the fluorescence signal,which was caused by N-methylmesoporphyrin IX(NMM)insertion into the G-quadruplex structure.This proposed strategy combines the excellent specificity between the aptamer and target,high sensitivity of the fluorescence signal by G-quadruplex and ExoⅢ-aided recycling amplification.We selected(50-1200 nmol/L)MUC1,a common tumor biomarker,as the proof-of-concept target to test the specificity of our aptasenso r.Results reveal that the sensor sensitively and selectively detected the target protein with limits of detection(LODs)of 3.68 and 12.83 nmol/L in buffer solution and 10%serum system,respectively.The strategy can be easily applied to other targets by simply substituting corresponding aptamers and has great potential in the diagnosis and monitoring of several diseases.