Micropipette tips

In this case, both the top and the bottom liquid phases contain the same ion at equilibrium. A micropipette tip is used to deplete concentration of this ion in the top solvent near the ITIES. This depletion results in the ion transfer across the ITIES (Fig. 13), which can produce positive feedback if the bottom phase contains a sufficiently high concentration of M ... 

The micropipette tip containing solid phases is a relatively new sample preparation technique that permits handling of microliter to submicroliter amounts of liquid samples, using the techniques of SPE, dialysis, and enzyme digestion. Various phases (reversed-phase, affinity, size-exclusion, etc.) are packed, embedded, or coated on the walls of pipette, permitting liquid samples to be transferred without undue pressure drop or plugging (Fig. 2.5). 

For most reactions, one can use 1-2 mM acetyl phosphate and 1-2 international units of acetate kinase. The enzyme is usually supplied as a crystalhne suspension in 3 M ammonium sulfate, and 10 mM ammonium ion is inhibitory. Therefore, a useful practice is to snip off 0.5 cm from a disposable Eppendorf micropipette tip to facih-tate removal of 10-20 microliters of the crystalline suspension then spin down the enzyme in a 1.5 ml disposable conical plastic centrifuge tube, and remove the ammonium sulfate solution with a wick of twisted Kim-wipe. The enzyme precipitate can now be taken up directly into your working buffer. Note Acetate kinase is inactivated by cold exposure, but incubation with 10 M ATP or GTP reactivates the enzyme if warmed to room temperature for 5-10 min. 

Reference and auxiliary electrodes are coupled in a micropipette tip. The reference electrode consists of an anodised silver wire introduced in a tip through a syringe rubber piston. The tip is filled with saturated KC1 solution and contains a low-resistance liquid junction. The platinum wire that acts as auxiliary electrode is fixed with insulating tape. For measurement recording the tip is fixed on an electrochemical cell Metrohm support allowing horizontal and vertical movement (see Fig. 36.1 of Procedure 36 in CD accompanying this book). 

UMEs used in our laboratory were constructed by sealing of carbon fibre into low viscosity epoxy resin (see Fig. 32.4) [118]. This method is simple, rapid and no specialised instrumentation is required. Firstly, the fibres are cleaned with this aim. They are immersed in dilute nitric acid (10%), rinsed with distilled water, soaked in acetone, rinsed again with distilled water and dried in an oven at 70°C. A single fibre is then inserted into a 100- iL standard micropipette tip to a distance of 2 cm. A small drop of low-viscosity epoxy resin (A. R. Spurr, California) is carefully applied to the tip of the micropipette. Capillary action pulls the epoxy resin, producing an adequate sealing. The assembly is placed horizontally in a rack and cured at 70°C for 8h to ensure complete polymerization of the resin. After that, the electric contact between the carbon fibre and a metallic wire or rod is made by back-filling the pipette with mercury or conductive epoxy resin. Finally, the micropipette tip is totally filled with epoxy resin to avoid the mobility of the external connection. Then, the carbon fibre UME is ready. An optional protective sheath can be incorporated to prevent electrode damage. 

The preparation of carbon disk-shape UMEs is similar to that of the carbon fibre UME. The only difference is that the active part is sealed on epoxy resin. With this purpose, a micropipette tip (1 mL) is glued to the head-tip of a carbon fibre UME. The carbon fibre is maintained in vertical position with a metal hook and the micropipette tip (1 mL) is filled with epoxy resin. Once the resin is cured, the tip is cross-sectioned with a microtome or a blade. Then, the disk carbon UME is ready. 

To make the reference electrode, anodize a 5-cm-long silver wire (1 mm diameter) in a saturated KC1 solution to form AgCl. Introduce the anodized wire in a 250 pL micropipette tip through a syringe rubber piston. Fill the tip, which contains a low-resistance liquid junction, with saturated KC1 solution. 

Cut 250 pL micropipette tips for obtaining 1-cm-long pieces with a diameter of 0.5 cm at the top. Adhere them concentrically to the chip holes (A, B, C, and D) with Araldit forming reservoirs of approximately 150 pL volume. The tip also allows fixing the platinum wire to the microchip. 

Further miniaturization of the SPE technique permits a reduction in the amount of organic solvent used, on-line coupling to analytical instruments, fast analysis times and excellent sensitivity. Downsizing of SPE has been focused mainly on the use of libers, beads, and adsorbents as extraction phases that are reproducibly packed in tubes, capillaries, syringes, needles, and even micropipette tips. 

One of the benefits of electrochemical batch injection analysis is that dilution of the sample with electrolyte is not necessary, see below. A sample of volume =sl00p.L is injected directly from a micropipette, tip diameter 0.5 mm, over the centre of a macroelectrode exactly as in a wall-jet system. This is equivalent to a flow injection system with zero dispersion. During the injection, and after a short initial period to reach steady-state, the hydrodynamics is wall-jet type and a time-independent current is registered. BIA was first devised using amperometric, e.g., [31], and potentiometric, e.g., [34], detection. A typical amperometric trace is shown in Fig. 16.5. By using a programmable, motorised electronic... 

Fig. 16.4. Modification of a large-volume wall-jet cell to a cell for batch injection analysis (BIA). Cell body of perspex, diameter 12cm A. disc electrode contact B, auxiliary electrode C, reference electrode D, micropipette tip.

Fig. 16.5. BIA chronoamperometric transient for the oxidation of 2mM lC Fe(CN)6 in 0.4 M K2S04 electrolyte injection of 100 p.L onto Pt disc electrode (d = 3.28 mm) at +0.6 V vs. SCE. Injection flow rate 75.3 (iL s micropipette tip internal diameter 0.47 mm. 1 = 0 corresponds to the start of the injection period.

Prepare separate tubes containing all of the sample chamber components of the desired assays of Series I through IV except for the addition of KCN solution where it is called for in Series III and IV. Add the components to the tubes in the order in which they are listed in the tables. Prevent plugging of micropipette tips used to transfer the heart particle suspension by cutting about 2 mm off the tips. 

Using a fresh micropipette tip, follow the same procedure (steps 6 and 7) to do twofold serial dilutions of the untreated IS sample across row B. Remember that well IB will contain no primary antibody. 

Micropipette tips may introduce contamination. If this is excessive it may be necessary to soak the tips in dilute nitric acid and then wash with high purity water before use. In any case, with each solution, the micropipette tip should be washed through twice with injections which are discarded into a beaker before it is used for the actual injection into the graphite tube. 

Centrifuge the cells at 3000x g for 5 min at room temperature. Resuspend the cells in 1 ml of sterile water. This should be done with great care because this step significantly influences the transformation efficiency. We normally cut off the tip of a 1-ml plastic micropipette tip and carefully pipette up and down a couple of times. 

One should not see the potentiometric mode as an alternative but as a complement to the amperometric mode. The potentiometric mode offers the possibility of performing measurements on non-redox active species, e.g., not detectable amperometrically in aqueous solutions. Another advantage is the increased selectivity of the potentiometric response compared to the Faradaic response. The logarithmic response is also useful when the species of interest is in low concentrations. Since the tip is passive, only substrate generation-tip detection experiments are possible. However, the tip does not alter the concentration profile of the chemically or electrochemically generated species. The potentiometric micropipette tips have very small dimensions, typically 1 /xm or less, and the shielding is minimal. 

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