Facilitated Ion Transfer Across the Micro-liquid/Liquid Interface Supported at the Tip of a Silanized Micropipette

Glass micropipettes with silanized inner walls can be filled with an organic solvent for voltammetric measurements in an aqueous solution. This arrangement was employed to investigate systematically the mechanism of facilitated potassium ion transfer by an ionophore dibenzo 18 crown 6(DB18C6) across a micro water/1,2 dichloroethane(W/DCE) interface supported at the tip of a silanized micropipette. Our experimental results verify that this facilitated ion transfer across the liquid/liquid interface did occur by an interfacial complexation dissociation process(TIC TID mechanism). The ratio of the diffusion coefficient of DB18C6 to that of its complexed ion in the DCE phase was calculated to be 1.74±0.07.

Mechanical Properties of Cells Estimated by Different Models of Micropipette Aspiration

Background The mechanical properties are related with many biological functions of cells. Accurate quantification of the mechanical properties of living cells require the combined use of experimental techniques and theoretical models. Micropipette aspiration (MPA) is one of common techniques in determining mechanical properties of the living cells. The halfspace model (HSM) is employed in MPA technique. However,in the conditions of linear constitutive relations and small deformations,the HSM is inadequate for characterizing the MPA of a spherical cell in two respects. Firstly,the cell size is fairly finite other than semi-infinite to the inner radius of a micropipette;Secondly,cells are compressible,with a Poisson’s ratioνvarying from 0. 2 to 0. 4 (23-25) instead of incompressible (ν=0. 5). Thus,a more accurate model is necessary.In this study,the viscoelastic expressions were derived from our previous MPA test. Then,a sphere model (SM) employed to analyze mechanical properties of rabbit chondrocytes combined with the experimental data. Differences in mechanical properties estimated by different mechanical models were evaluated.Methods A sphere model (SM) was employed. The relative dimension of cell to micropipette and the compressibility of the cell were taken into account,as shown in Fig. 1a.■Fig.1 Sphere model of the MPA of a single cell employing different constitutive relationships The approximate expression for the aspirated length was obtained from our previous study as follows:■Furthermore,assuming that the cell behaves as a homogeneous and isotropic standard linear solid (Fig. 1b),two viscoelastic creep expressions of the aspirated length for incompressible sphere model (ICSM) and for compressible sphere model (CSM) were derived by elastic-viscoelastic correspondence principle and integral transformation as Eqs.(2) and (3)respectively.■Results(1) Comparisons of models The elastic modulus from the ICSM was 47. 4%higher than that of the half-space model (HSM)(P<0. 001). For the CSM,the percentage increase in E over the value for the HSM was 87. 7%,78. 9%,and 64. 9%when the Poisson’s ratio was set to 0. 2,0. 3,and 0. 4,respectively.For the viscoelasticity,the parameters for the ICSM and CSM were significantly larger than those of the HSM (P <0. 001). The k1,k2,andμfor the ICSM were 37. 8%,37. 9%,and 39. 0%higher,respectively,than those of the HSM. For the CSM,the viscoelastic parameters decreased with the increase ofν. Whenν=0. 3,k1,k2,andμincreased by 71. 0%,200%,and 157%,respectively,compared to those of the ICSM (P<0. 001);For the cases ofν=0. 2 andν=0. 4,the above parameters were respectively 102%,243%,and 209%and 35. 3%,97. 5%,and 79%higher than those of the ICSM.(2) Predictions for the relative errors of mechanical parameters caused by HSM e is defined as the relative change of elastic moduli (or relative error) between the HSM and SM. As shown in Fig. 2,when Poisson’s ratioνis 0. 3,in order to let the e less than 30%,relative dimension between the cell and the micropipetteξneeds to be at least 5. 0. Whenνequals 0. 5 (ICSM),ξis about 3. 3 to make the e reach 20%. However,ξis rarely larger than 5. 0 in general MPA experiments,thus the relative error of modulus will exceed 30%. The above results are independent of cell types,thus they are applicable to other spherical solid-like cells.■Fig.2 Thresholds ofξvarying withνwhen e was 10%,20%,and 30%,respectively Another parameter VR was introduced to represent the relative errors of viscoelastic parameters between HSM and SM.With regard to ICSM (Fig. 3a),whenξis 3,VRis nearly 22%. If theξis larger than 8. 0,the relative error will be reduced to less than 10%. For the CSM,the viscoelastic parameters of a typical chondrocyte varying withξandνwere obtained,as shown from Figs. 3b to 3d. Whenνtends to 0. 5,the parameters tend to those of ICSM. When theξexceeds 10,each parameter changes very little. For a certain Poisson’s ratio (ν=0. 3),whenξis 3,the VR of k1,k2,andμare 47. 1%,70. 8%,and 68. 2%,respectively. Whenξequals 5 and 10,the above values are 42. 3%,68. 8%,65%,and 38. 4%,66. 0%,63. 2%,respectively. For a givenξ(ξ=3),whenνis 0. 2,the VR of k1,k2,andμare 53. 6%,73. 3%,and 75. 0%,respectively.Whenνis taken as 0. 3 and 0. 4,the above errors are 47. 7%,71. 1%,68. 2%,and 38. 4%,58. 8%,54. 8%,respectively.Thus,the VR also decreases with the increase ofξandν.Conclusions The effects of the relative dimension between the cell,and micropipette and the Poisson’s ratio of cell were remarkable and should be taken into consideration in the pursuit of more accurate mechanical parameters of cells.

Modeling of micropipette aspiration and optical tweezers stretching of erythrocytes with or without Malaria parasite

The erythrocytes play an important role in the human body. The healthy erythrocytes can undergo extremely large deformation while passing through small capillaries. Their infection by Malaria Plasmodium falcipurum （P.f.） will lead to capillary blockage and blood flow obstruction. Many experimental and computational methods have been applied to study the increase in stickiness and decrease in deformability of the Malaria （P.f.） infected erythrocytes. The novelty of this paper lies in the establishment of an multi-component model for investigating mechanical properties of Malaria （P.f.） infected erythrocytes, especially of their enclosed parasites. Finite element method was applied to simulate the erythrocytes＇ deformation in micropipette aspiration and optical tweezers stretching using the computational software ABAQUS. The comparisons between simulations and experiments were able to quantitatively conclude the effects of stiffness and stickiness of the parasitophorous vacuole membrane on the cells＇ deformation, which could not be obtained from experiments directly.

Mechanisms of sodium and potassium ions transfer facilitated by dibenzo-15-crown-5 across the water/1,2-dichloro-ethane interface using micropipettes

The transfer of sodium and potassium ions facilitated by dibenzo-15-crown-5 (DB15C5) has been studied at the micro-water/l,2-dichloroethane (water/DCE) interface supported at the tip of a micropipette. Cyclic volt-ammetric measurements were performed in two limiting conditions: the bulk concentration of Na+ or K+ in the aqueous phase is much higher than that of DB15C5 in the organic phase (DB15C5 diffusion controlled process) and the reverse condition (metal ion diffusion controlled process). The mechanisms of the facilitated Na+ transfer by DB15C5 are both transfer by interfacial complexation (TIC) with 1:1 stoichiometry under these two conditions, and the corresponding association constants were determined at log β1 = 8.97±0.05 or log β1 = 8.63±0.03. However, the transfers of K+ facilitated by DB15C5 show different behavior. In the former case it is a TIC process and its stoichiometry is 1:2, whereas in the latter case two peaks during the forward scan were observed, the first of which was

A study of the initial adhesive force of cells on silk fibroin-based materials using micropipette aspiration

With the development of biomaterials,more attention is paid to the adhesion characteristics between cells and materials.It is necessary to study the adhesive force with a suitable method.Silk fibroin(SF)is widely investigated in biomedical application due to its novel biocompatibility and mechanical properties.In this article,the micropipette aspiration method and measurement pattern of uniform cells in round shape(UCR)was used to study the initial adhesive force of three types of cells on pure silk fibroin films(SFFs).We also compared the adhesive forces of modified SFFs with that of pure SFFs.The results of adhesive force in the initial adhesive stage were in concordance with the results of MTT assay andmicroscope observation,which were confirmed by the above three cell lines and four kinds of SFFs.The results indicated UCR was an efficient and quantitative measurement pattern in initial adhesion stage.This article also provides a useful method in identifying initial cell-materials interactions.

Design of Micropipette System with High Precision for Small Enzyme Immunoassay Analyzer

A small auto micropipette system is developed to improve the reliability and accuracy of the automatic enzyme immunoassay analyzer’s microscale pipetting system. A sophisticated injection mechanism is designed by the means of dislocation parallel distribution of the screw and injector piston rod. It possesses the function of pipetting, taking and removing the pipette tips. In the control system, STM32 controller is used, controlling the single-axis S-type acceleration/deceleration algorithm and multi-threaded coordinated motion. The acceleration/deceleration curves are analyzed and optimized by using the method of segmentation;a minimum injection rate of 1 μL and a step rate of 0.05 μL are realized. The method of digital image processing is used to detect the amount of pipetting in micro-pipetting quantitatively. The liquid area is extracted by background contrast method, and the liquid volume in the tip is obtained by combining the geometric characteristics of the disposable tip, when the pipetting capacity is not qualified to carry out specific guidance on the pipetting system, and avoid the blocking needle, bubble and other abnormal pipetting phenomenon on the impact of pipetting accuracy. The experimental results show that the combination of the automatic sampling system and the image flow detection system can effectively improve the precision and reliability of the micro-pipetting system. Finally, the injection accuracy of the system at the test points with 10, 50 and 100 μL liquid volumes reaches 1.8%, 1.28% and 1.15%respectively.

GMP法玻璃化冷冻人胚胎的效果观察

目的探索GMP法玻璃化冷人胚胎的可行性。方法选用IVF中多精受精的4-10细胞阶段的胚胎为研究对象，先后在玻璃化1液（10％乙二醇＋10％二甲亚砜）和玻璃化2液（20％乙二醇＋20％二甲亚砜＋0．3M蔗糖）中分别平衡1mim和0．5mim，然后用GMP管虹吸胚胎直接浸入液氮。其结果与常规的程序化慢冻进行比较。结果GMP法的存活率、胚胎完整率、卵裂球存活率比程序化慢冻法更高（分别为94．4％对73．9％，P〈0．01；79．6％对33．9％，P〈0．01；91．6％对67．8％；P〈0．01），但卵裂球完全死亡率更低（0％对10、4％；P〈0．01）。结论GMP法玻璃化冷冻人胚胎是可行的。

Integrin betal mediates hepatocellular carcinoma cells chemotaxis to laminin

BACKGROUND: Chemotaxis is an important step durin the invasion of carcinoma cells. And integrins are most im portant receptors mediating interaction between cells an extracellular matrix ( ECM). This study was designed t study integrin betal mediating chemotaxis of hepatocellula carcinoma (HCC) cells to laminin (LN). METHODS: A micropipette technique was adopted to in vestigate the effect of blockade of integrin betal on pseudo pod protrusion of HCC cells in response to LN stimulation Chemotactic pseudopod protrusion of a HCC cell was eva luated using a dual-pipette set-up, in which two pipette filled with LN solution were positioned in close contact wit the same cell, and pseudopod protrusion into each pipett was viewed dynamically and recorded with a tape recorder The lengths of pseudopods were measured and plotte against time to obtain a pseudopod growth curve. The in tegrin betal subunit on the surfaces of HCC cells were ana lyzed by flow cytometry. RESULTS: In dual pipette chemotaxis experiment, whe the two pipettes were filled with LN(50 μg/ml, 200 μg/ml) pseudopods extended from the HCC cell into each of th pipettes nearly symmetrically, ie, with nearly identica maximum pseudopod length and similar pseudopod growth curves. Upon addition of anti-CD29 (20 μg/ml) to one o the pipettes, pseudopod protrusion was blocked nearly completely while protrusion into the opposite pipette be came more evidently, with a larger maximum length. Ex pression of integrin betal was up to 95.78% to cells chosen in the experiment. CONCLUSION: Integrin betal subunit was an importan constituent receptor subunit for mediating chemotactic pseudopod protrusion of HCC cell to LN.

Adhesive properties of hepatoma cells to collagen Ⅳ coated surfaces

Objectives: To quantitatively study the adhesive pro- perties of hepatoma cells to collagen Ⅳ coated artifi- cial basement membrane and to investigate the rele- vance of cell adhesive forces to the concentration of collagen Ⅳ. Methods: Synchronous G1 and S phase cells were a- chieved using thymine-2-desoxyriboside and cochicine sequential blockage method and double thymine-2- desoxyriboside blockage method respectively. The adhesive forces of hepatoma cells were investigated by micropipette aspiration technique. Results: The adhesive forces of hepatoma cells to ar- tificial basement membrane were (107.78±65.44) ×10^(-10)N, (182.60±107.88)×10^(-10)N, (298.91± 144.13)×10^(-10)N when the concentration of the membrane coated by 1, 2, 5μg/ml collagen Ⅳ re- spectively (P<0.001). The adhesive forces of G1 and S phases hepatoma cells to artificial basement membrane were (275.86±232.80)×10^(-10)N and (161.16±120.40)×10^(-10)N respectively when the concentration of the membrane coated by 5μg/ml collagen Ⅳ (P<0.001). Conclusions: The adhesive forces of hepatoma cells to artifical basement membrane in direct proportion to the concentration of collagen Ⅳ suggests that the in- crease of basement membrane might be conducive to the chemotactic motion and adhesiveness of tumor cells. G1 phase cells are more capable of adhering to basement membrane than S phase cells. Hepatoma cells, especially G1 phase cells, may survive in blood circulation, and sequest and adhere in microcircula- tion, and get through basement membrane for re- mote metastasis.

Normal age-related viscoelastic properties of chondrons and chondrocytes isolated from rabbit knee

Background The mechanical microenvironment of the chondrocytes plays an important role in cartilage homeostasis and in the health of the joint. The pericellular matrix, cellular membrane of the chondrocytes, and their cytoskeletal structures are key elements in the mechanical environment. The aims of this study are to measure the viscoelastic properties of isolated chondrons and chondrocytes from rabbit knee cartilage using micropipette aspiration and to determine the effect of aging on these properties. Methods Three age groups of rabbit knees were evaluated： （1） young （2 months, n=10）; （2） adult （8 months, n=10）; and （3） old （31 months, n=10）. Chondrocytes were isolated from the right knee cartilage and chondrons were isolated from left knees using enzymatic methods. Micropipette aspiration combined with a standard linear viscoelastic solid model was used to quantify changes in the viscoelastic properties of chondrons and chondrocytes within 2 hours of isolation. The morphology and structure of isolated chondrons were evaluated by optical microscope using hematoxylin and eosin staining and collagen-6 immunofluorescence staining. Results In response to an applied constant 0.3-0.4 kPa of negative pressure, all chondrocytes exhibited standard linear viscoelastic solid properties. Model predictions of the creep data showed that the average equilibrium modulus （E~）, instantaneous modulus （E0）, and apparent viscosity （~） of old chondrocytes was significantly lower than the young and adult chondrocytes （P 〈0.001）; however, no difference was found between young and adult chondrocytes （P 〉0.05）. The adult and old chondrons generally possessed a thicker pericellular matrix （PCM） with more enclosed cells. The young and adult chondrons exhibited the same viscoelastic creep behavior under a greater applied pressure （1.0-1.1 kPa） without the deformation seen in the old chondrons. The viscoelastic properties （E,, E0, and/~） of young and adult chondrons were significantly greater than that observed in young and adult cells, respectively （P 〈0.001）. The adult chondrons were stiffer than the young chondrons under micropipette aspiration （P 〈0.001）. Conclusions Our findings provide a theoretical model to measure the viscoelastic properties of the chondrons as a whole unit by micropipette aspiration, and further suggest that the properties of the chondrocytes and PCM have an important influence on the biomechanical microenvironment of the knee joint cartilage degeneration that occurs with aging.

Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL

Using the method of gene transfection with liposome, we obtained the mouse erythro-leukemia cell line MEL-TF19, which stably carries TFAR19, a novel apoptosis-related gene. The expression of TFAR19 was detected by Western blot. Growth curve and flow cytometry analysis showed that after being transfected with TFAR19 gene, the growth of MEL-TF19 is suppressed and its apoptosis is accelerated because of the serum deprivation. Our biorheological study indi-cated that in the apoptotic process, compared with MEL cells, MEL-TF19 cells exhibit larger os-motic fragility, lower cell surface charge density, increased elastic modulus K1 which is inversely proportional to cells?maximal deformation ability, obviously diminished surface viscosity m, with elastic modulus K2 having no distinct changes. The above results provided some bases for recog-nizing the function of TFAR19 completely from the viewpoint of biorheology.

ADHESION STRENGTH AND MORPHOLOGIES OF r BMSCs DURING INITIAL ADHESION AND SPREADING

Cell adhesion plays an important role in cell physiology. A better understanding of this process could facilitate many clinical therapies. In this study, Rat bone marrow-derived mesenchymal stem cells （rBMSCs） were cultured on glass substrate, and the morphology and adhesion strength were characterized. The cell morphology was defined as spherical, adhesive, and spreading. The adhesion strengths of the different morphologies exhibited different distribu- tion patterns. The spherical cells exhibited low adhesion strength; the adhesive cells exhibited rapidly increasing adhesion strength while their diameters remained relatively constant. The ad- hesion strength increased with the cell diameter in the spreading cells. These findings suggest that adhesion strength can be quickly assessed by examining the cell morphology.

Ion current rectification: from nanoscale to microscale

Ion current rectification(ICR)is an electrodynamic phenomenon in electrolyte solution which is defined as the asymmetric potential-dependent ion flux through a confined environment,giving rise to asymmetric electrical current-voltage characteristics induced by the influence of an asymmetric electrical double layer structure.Since the discovery of the ICR phenomenon,the observation and application of ICR at nanoscale and microscale have been widely investigated experimentally and theoretically.Here,the recent progress of ICR from nanoscale to microscale is systematically reviewed.Nano/micropore structures of different materials,shapes and pore sizes are first discussed.Then,the factors influencing ICRs by thermodynamically or kinetically regulating the electrical double layer structure are introduced.Moreover,theoretical models are presented to explain the mechanism of ICRs.Based on the understanding of this phenomenon,the applications,especially in biosensors,are discussed.Finally,future developments of this area are briefly presented.This review covers the representative related literature published since 2010 and is intended to give a systematic introduction to this area.