Gel-loading Tip Research Articles

Rapid analysis of untreated food samples by gel loading tip spray ionization mass spectrometry.

Rapid, efficient, versatile, easy-to-use, and non-expensive analytical approaches are globally demanded for food analysis. Many ambient ionization approaches based on electrospray ionization (ESI) have been developed recently for the rapid molecular characterization of food products. However, those approaches mainly suffer from insufficient signal duration for comprehensive chemical characterization by tandem MS analysis. Here, a commercially available disposable gel loading tip is used as a low-cost emitter for the direct ionization of untreated food samples. The most important advantages of our approach include high stability, and durability of the signal (> 10min), low cost (ca. 0.1 USD per run), low sample and solvent consumption, prevention of tip clogging and discharge, operational simplicity, and potential for automation. Quantitative analysis of sulfapyridine, HMF (hydroxymethylfurfural), and chloramphenicol in real sample shows the limit-of-detection 0.1μgmL-1, 0.005μgmL-1, 0.01μgmL-1; the linearity range 0.1-5μgmL-1, 0.005-0.25μgmL-1, 0.01-1μgmL-1; and the linear fits R2 ≥ 0.980, 0.991, 0.986. Moreover, we show that tip-ESI can also afford sequential molecular ionization of untreated viscous samples, which is difficult to achieve by conventional ESI. We conclude that tip-ESI-MS is a versatile analytical approach for the rapid chemical analysis of untreated food samples.

High ohmic resistor hyphenated gel loading tip nano-electrospray ionization source for mini mass spectrometer

The deployment of mini mass spectrometers on the field strongly demands efficient ionization sources that are easy-to-operate. Nano-electrospray (nESI) ion source has been widely used in the field of chemistry, biology, medicine, pharmaceutical industry, clinical assessment and forensic science. In this study, a high ohmic resistor hyphenated gel loading tip nESI source was coupled with our home developed mini mass spectrometer. This ionization source has the advantages of simple-in-design, disposable and low-in-cost, therefore it could be frequently used for analysis of aqueous samples without leading to cross contamination. Performances of the gel loading tip nESI emitter were similar to pulled glass capillary, and highly compatible for the analysis of biomolecule in aqueous solution. Different peptide and small molecules have been confirmed with a continuous atmospheric pressure-interfaced (CAPI) mini mass spectrometer. The corona discharge, which was usually observed at nESI emitter tip under high aqueous solvent conditions, resulting in low ion intensity, has been successfully quenched using a 10 GΩ resistor in both a pulled glass capillary and a gel loading tip as nESI emitter in this study. Compared with conventional ESI, the metal wire assisted gel loading tip facilitated loading and direct analysis of biological samples without sample pretreatment.

Remote sampling mass spectrometry for dry samples: Sheath-flow probe electrospray ionization (PESI) using a gel-loading tip inserted with an acupuncture needle.

Probe electrospray ionization (PESI) is only applicable to liquid or wet samples. In this study, a sheath-flow PESI method for remote sampling mass spectrometry that can be applied to dry samples was developed. An acupuncture needle (0.12mm outer diameter, 700nm tip diameter) was inserted into a gel-loading tip with a 0.1mm protrusion out of the tip. Analytes were extracted by filling the latter tip with solvent and softly touching the sample surface for a short time (<1s). A high voltage was applied to the acupuncture needle, and mass spectra of analytes were obtained by self-aspirating electrospray. Dry samples, such as lines of ballpoint pen ink on paper, pharmaceutical tablets, instant coffee, brown rice, and narcotics, gave strong ion signals. The sample carryover was negligible. The sequential electrospray was observed to be similar to conventional PESI. The limits of detection (LODs) for morphine and rhodamine B were found to be of the order of picograms. Because of its simplicity and versatility, sheath-flow PESI is a promising technique for on-site and nondestructive profile analysis of dry samples with bulky and complicated shapes, with a spatial resolution of ~0.3mm.

A method of islet transplantation under kidney capsule in mouse and the evaluation of it's effect

Objective In this paper, we introduced a simple and easy way to do islet transplantation under kidney capsule in mouse, and the effect evaluation of it’s blood glucose, oral glucose tolerance and histological stain after transplantation. Methods BALB/c mice are used in the experiment.The islets were harvested by reversely perfusion of collagenase V into common bile duct and purification by Histopaque density gradient followed by manual picking. After a simple culture, the islets were transplanted under the kidney capsule of diabetic BALB/c mice using a gel loading tip. Then the blood glucose and the oral glucose tolerance test were tested regularly.The kidney was cut at the 10th day after the islet transplantation.The HE and immunofluorescence staining of islet under kidney capsule were also done to evaluate the islet morphology and function. Results We get 1 063± 122 islets, or 1 259.72±107.19 IEQ from four mice at one time.The level of blood glusoce turned down to normal in 24 h after the islet transplantation.Oral glucose tolerance test(OGTT) results showed that the blood glucose levels of mice after islet transplantation were significantly lower than those of diabetes(P< 0.01).And the results of immunofluorescence staining showed that the activity of islets was good[(92.8±2.6)%]. Conclusion The method of the experiment is simple, and the islet activity is good after transplantation, which make a good foundation for the further research on drug screening for islet transplantation. Key words: BALB/c mouse; Islet transplantation; Kidney capsule; Diabetes

Realizing nano electrospray ionization using disposable pipette tips under super atmospheric pressure

Nano electrospray with a solution flow rate below 100 nL min(-1) is usually performed using a metallized pulled glass capillary with an inner diameter tip of <10 μm. In this paper, we demonstrated that it was possible to achieve a stable solution flow rate of ~10 nL min(-1) for an aqueous solution using a disposable plastic pipette tip with an inner diameter of 100 μm as an ESI emitter. This pipette tip (gel loading tip) was originally designed for the loading of sample onto the gel in gel electrophoresis. The inner and outer diameters were much larger than those of a nanoESI emitter, but the low solution flow rate could be sustained by conducting the electrospray under a super-atmospheric gas pressure. The high pressure condition ensured the absence of electrical discharge which was essential for stable electrospray of aqueous solution. ESI with this low cost tip was equally as sensitive as the standard nanoESI emitter and capable of producing a long lasting signal from a small amount of sample liquid. Due to a small initial droplet size under the low flow rate regime, ESI with the present method exhibited the property of nanoESI where the ion suppression of the hydrophilic analyte by the hydrophobic compound present in the solution was greatly reduced. Furthermore, owing to its larger inner diameter, the tip relatively easily avoided the clogging problem often encountered when dealing with "dirty" samples when compared with a nanoESI emitter made from a fine glass capillary.

Biomolecular Analysis and Biological Tissue Diagnostics by Electrospray Ionization with a Metal Wire Inserted Gel-Loading Tip

A metal wire-inserted disposable gel-loading tip was examined as an electrospray emitter. Its performance was similar to that of conventional electrospray ionization (ESI) with a relatively low flow rate (~100 nL/min) and without the need for solvent pumps. It was also used as an emitter for solid probe-assisted ESI (SPA-ESI) (e.g., biofluid was sampled from the biological tissue by a needle and was inserted into the solvent-preloaded gel-loading tip). Selective detection of lipids and proteins, such α and β chains of hemoglobin could be accomplished by choosing appropriate solvents. A suitable protocol for cancer diagnosis was established by this method. A good figure of merit of this method is its applicability to biological tissue diagnostics with high cost efficiency and on a disposable basis.

Identification of azurocidin as a potential periodontitis biomarker by a proteomic analysis of gingival crevicular fluid

BackgroundThe inflammatory disease periodontitis results in tooth loss and can even lead to diseases of the whole body if not treated. Gingival crevicular fluid (GCF) reflects the condition of the gingiva and contains proteins transuded from serum or cells at inflamed sites. In this study, we aimed to discover potential protein biomarkers for periodontitis in GCF proteome using LC-MS/MS.ResultsWe identified 305 proteins from GCF of healthy individuals and periodontitis patients collected using a sterile gel loading tip by ESI-MS/MS coupled to nano-LC. Among these proteins, about 45 proteins were differentially expressed in the GCF proteome of moderate periodontitis patients when compared to the healthy individuals. We first identified azurocidin in the GCF, but not the saliva, as an upregulated protein in the periodontitis patients and verified its increased expression during periodontitis by ELISA using the GCF of the classified periodontitis patients compared to the healthy individuals. In addition, we found that azurocidin inhibited the differentiation of bone marrow-derived macrophages to osteoclasts.ConclusionsOur results show that GCF collection using a gel loading tip and subsequent LC-MS/MS analysis following 1D-PAGE proteomic separation are effective for the analysis of the GCF proteome. Our current results also suggest that azurocidin could be a potential biomarker candidate for the early detection of inflammatory periodontal destruction by gingivitis and some chronic periodontitis. Our data also suggest that azurocidin may have an inhibitory role in osteoclast differentiation and, thus, a protective role in alveolar bone loss during the early stages of periodontitis.

Gel-Loading Tip Vitrification of In Vitro-Matured Bovine Oocytes and Subsequent Embryo Production by IVF and Nuclear Transfer

The purpose of this study was to cryopreserve bovine oocytes for subsequent blastocyst production by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). A vitrification procedure using gel-loading tips as containers was applied to cryopreserve in vitro-matured and denuded oocytes. In Experiment 1, oocytes were vitrified-warmed in vitrification solution (VS) containing 25, 28, 31, or 40% ethylene glycol (EG) and 1.0 M sucrose. The proportions of survived oocytes that appeared to be morphologically normal after warming, and cleaved oocytes after IVF were lower with 25% EG-based VS when compared with 28–40% EG-based VS. Blastocyst yields 8 days after IVF of oocytes vitrified-warmed in 28 and 31% EG-based VS (12 and 17%, respectively) were not significantly different from those of the fresh control group (32%). Day-7 blastocysts derived from vitrified oocytes were composed of a smaller number of inner cell mass (ICM) and trophectoderm cells than the fresh Day-7 blastocysts. In Experiment 2, oocytes vitrified-warmed in 31% EG-based VS were subjected to enucleation and SCNT. The proportions of oocytes fused and cleaved in the vitrified group were comparable to those in the fresh control group. Blastocyst yields 6 and 7 days after SCNT of vitrified oocytes were lower than those of control oocytes, but 8 days after the SCNT, the difference became statistically comparable (45 versus 58% in control group). ICM and trophectoderm cell numbers in Day-7 blastocysts derived from vitrified oocytes were smaller than those of control blastocysts due to a slower developmental rate. In conclusion, bovine oocytes cryopreserved by vitrification in gel-loading tip were capable of developing into blastocysts after conventional IVF and SCNT, with slightly smaller cell numbers and a slower developmental rate.

Identification of a Cooperative Mechanism Involving Interleukin-13 and Eotaxin-2 in Experimental Allergic Lung Inflammation

Pulmonary eosinophilia, a hallmark pathologic feature of allergic lung disease, is regulated by interleukin-13 (IL-13) as well as the eotaxin chemokines, but the specific role of these cytokines and their cooperative interaction are only partially understood. First, we elucidated the essential role of IL-13 in the induction of the eotaxins by comparing IL-13 gene-targeted mice with wild type control mice by using an ovalbumin-induced model of allergic airway inflammation. Notably, ovalbumin-induced expressions of eotaxin-1 and eotaxin-2 mRNA in the lungs were almost completely dependent upon IL-13. Second, in order to address the specific role of eotaxin-2 in IL-13-induced pulmonary eosinophilia, we generated eotaxin-2 gene-deficient mice by homologous recombination. Notably, in contrast to observations made in eotaxin-1-deficient mice, eotaxin-2-deficient mice had normal base-line eosinophil levels in the hematopoietic tissues and gastrointestinal tract. However, following intratracheal IL-13 administration, eotaxin-2-deficient mice showed a profound reduction in airway eosinophilia compared with wild type mice. Most interestingly, the level of peribronchial lung tissue eosinophils in IL-13-treated eotaxin-2-deficient mice was indistinguishable from wild type mice. Furthermore, IL-13 lung transgenic mice genetically engineered to be deficient in eotaxin-2 had a marked reduction of luminal eosinophils. Mechanistic analysis identified IL13-induced eotaxin-2 expression by macrophages in a distinct lung compartment (luminal inflammatory cells) compared with eotaxin-1, which was expressed solely in the tissue. Taken together, these results demonstrate a cooperative mechanism between IL-13 and eotaxin-2. In particular, IL-13 mediates allergen-induced eotaxin-2 expression, and eotaxin-2 mediates IL-13-induced airway eosinophilia.

Improved survival of vitrified in vivo-derived porcine embryos.

An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use.

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80–93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.

Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes

Polyvinyl alcohol (PVA) was used as a substitute for serum in a vitrification solution for in vitro matured bovine oocytes. In vitro matured bovine oocytes were cryopreserved in various vitrification solutions (VS) supplemented with different concentrations (0.05, 0.1, 0.5, and 1%) of PVA, 20% fetal calf serum (FCS) or without macromolecule supplementation in a gel-loading tip (GL-tip). After warming, vitrified oocytes were examined for effects on survivability, fertilizability, and embryonic development in vitro. At 18 h in vitro fertilization after vitrifying and warming, the number of surviving mature oocytes vitrified in VS without macromolecule supplementation was significantly ( P<0.05) lower than those with macromolecule supplementation. For fertilizability after vitrification, there was no significant difference in the penetration rate of oocytes among fresh oocytes (98.7%); oocytes vitrified in VS supplemented with 0.1 (76.8%), 0.5 (70.2%), or 1% (80.3%) PVA; 20% (84.1%) FCS; or without supplementation (61.7%). Also, the normal fertilization rate was not significantly different in oocytes vitrified with 0.1 (56.5%), 0.5 (43.5%), or 1% (49.7%) PVA and 20% (60.6%) FCS, compared with fresh oocytes (84.0%). Subsequently, vitrified oocytes were examined for embryonic development effects in vitro. The highest proportion of cleaved oocytes after vitrification was obtained in VS supplemented with 0.1% (18.8%) PVA. Additionally, the proportion of development to morula stage (7.7%) in the oocytes vitrified in a VS supplemented with 0.1% PVA was significantly ( P<0.05) superior to that of the 0, 0.5, and 1% PVA-vitrified groups. However, the beneficial effect of PVA addition was not found in blastocyst development. Embryonic development of vitrified oocytes was significantly lower than that of fresh oocytes. In conclusion, the present results indicate that 0.1% PVA supplementation in VS results in a significantly higher rate of morula stage embryos than 0, 0.5, and 1% PVA supplementation, and could replace FCS in VS for vitrification of in vitro matured bovine oocytes.

Gel-Loading Tip As Container for Vitrification of In Vitro-Produced Bovine Embryos.

Recently, new vitrification methods for bovine embryos characterized by ultra-rapid cooling have been reported. The present study was undertaken to determine the availability of gel-loading tips as containers for vitrification of in vitro-produced bovine embryos at various developmental stages. Bovine embryos at Days-1, -2, -3, -4, -5, and -7 (the day of insemination was defined as Day-0) were placed in 0.6~0.7 μl of vitrification solution (20% ethylene glycol, 20% DMSO and 0.6 M sucrose) in a commercially available gel-loading tip, and cooled by immersing in liquid nitrogen. Survival of the vitrified embryos was evaluated by in vitro development into the blastocyst stage. There was no significant difference (P ≥0.05) between the survival rates for fresh and vitrified embryos at Day-1 (70.5 and 58.0%, respectively), Day-2 (75.9 and 68.3%, respectively), Day-3 (75.9 and 57.9%, respectively), Day-4 (80.0 and 67.6%, respectively), Day-5 (82.1 and 60.0%, respectively) and Day-7 (100 and 97.8%, respectively). And no significant differences were found between the total numbers of cells or the proportion of inner cell mass cells of blastocysts developed from fresh versus vitrified embryos. These results demonstrate that the gel-loading tip is applicable as a container for vitrification of in vitro-produced bovine embryos at the 2-cell to blastocyst stages.

Effect of β-mercaptoethanol on developmental competence of fair quality bovine embryos following culture and freezing

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80–93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.