Extraction, solid phase using beads

Figure 8.13. Solid-phase reversible immobilization (SPRI) extraction of genomic DNA from blood using magnetic silica beads.

With the need to provide PCR-amplifiable DNA, multiple approaches for incorporation of the extraction protocol onto microchips were examined. Recent development includes the implementation of a solid-phase extraction of DNA on a microchip [49]. The extraction procedure utilized was based on adsorption of the DNA onto bare silica. The silica beads were immobilized into the channel using a sol-gel network. This method made possible the extraction and elution of DNA in a pressure-driven system. 

Other than beads, porous polymer monoliths, which were photopolymerized in a COC chip, were used for solid-phase extraction. It is known that priming polymeric surfaces is not as simple as priming silica surfaces, which use a common surface primer agent, TMPM. Therefore, the grafting method as initiated by UV should be used to attach the polymer monoliths [588]. A similar strategy was used for sample pre-concentration of PAHs (e.g., pyrene). Pyrene (900 nM) was first concentrated by 400-fold in 24% ACN before switching to 56% ACN for CEC separation (see Figure 5.5) [148]. 

DNA extraction and purification were traditionally accomplished using organic extraction and ultracentrifugation-based procedures, which are both time-consuming and not easily transferable to the microscale. Newer methods employ solid-phase extraction (SPE) on silica surfaces, glass fibers, modified magnetic beads, and ion-exchange resins—techniques that save time and are also more amenable to chip applications. 

Numerous articles can be found on the use of sorbent materials such as silica, stainless steel beads, and common solid-phase extraction materials for collection of supercritical fluid-extracted analytes.38 2 In this scenario the fluid is usually allowed to expand into a gas, which is forced through a packed bed of solid sorbent material. The analytes are bound or simply deposited and cryogenically trapped onto the sorbent and are later eluted with appropriate solvents. The advantage of this technique is that further cleanup can be performed by carefully selecting the eluting solvent. 

One recent application in lEC consists of solid phase extraction disks which are used to eliminate matrix interference prior to analysis. The disk contains a membrane composed of resin beads enmeshed in a PTFE membrane, housed in a polypropylene housing. The resin is treated to remove specific ions such as H+ to remove hydroxide, Ag+ for excess halides and Ba to remove sulphate. Other disks such as hydroxide for acid removal and neutral styrene-divinylbenzene to eliminate hydrophobic components which can damage resin based IC columns are being developed [23]. 

Besides choosing the appropriate Ab, the assay method can be designed or manipulated to improve assay specificity using (a) protein precipitation, (b) liquid/liquid or solid-phase extraction, (c) HPLC separation of the analyte from the interfering compounds, (d) sample dilution with buffer or control matrix, or (e) an affinity solid phase (e.g., antibody-coated microtiter plate or polystyrene beads) to capture the analyte followed by wash steps. Affinity-purified antibodies and protein blockers are used in EIA to decrease nonspecific binding in plate assays. Increasing incubation time to reach equilibrium also improves binding specificity. 

One application example on the microfluidic LSI platform is the extraction of nucleic acids (NA) from a small amount of cells [126, 127] for cell-based assays. For the extraction of NA from a cell suspension, the cell membrane has to be destroyed first (chemical lysis of the cell). Afterwards, the NA are specifically separated from the residual cell components using a solid phase extraction method based on an NA affinity column (paramagnetic beads). This extraction protocol is completely implemented on the microfiuidic... 

The relative PLMA MW was determined by using nonaqueous SEC with THF as solvent and an HP1037A refractive index detector from Hewlett-Packard. A solid-phase extraction cartridge (Waters Sep-Pak) was used to take the PLMA and surfactants from the water phase to THF and to adjust the concentration for SEC. Sodium sulfate was used to dry the THF extract before injection into the SEC columns. For some samples there was too much surfactant present, and it overloaded the cartridge in such cases ion-exchange beads were used to reduce the concentration of the ionic surfactants in the aqueous phase before extraction. The advantage of this extraction procedure is its simplicity. The overall separation scheme is shown in Scheme I. 

In 1990, a solid-phase immunobead assay (SPIA), with colored polystyrene particles coated with anti-CTX monoclonal antibody (MAb-CTX) began to be used for direct detection of ciguatoxins adsorbed on bamboo paddles coated with organic correction fluid. The membrane immunob-ead assay (MIA) presented by in 1998 Hokama et al. is based on the immunological principles used to develop the SPIA, using a MAb-CTX coated onto colored polystyrene beads. The polyether toxins extracted from a piece of fish tissue bind to the hydrophobic membrane on a plastic support (membrane stick), and can be detected with the MAb-CTX coated onto the colored polystyrene beads. The color intensity of the membrane is related to the concentration of the toxin bound to the membrane. 

Since the last decade, the hypercrosslinked polystyrene sorbents have also come into routine use in analytical chemistry. They are widely used as excellent solid-phase extraction (SPE) media for the pre-concentration of trace amounts of organic contaminants in the environment, food products, biological fluids, gases, aquatic pools, etc., in combination with various chromatographic analytical techniques. By now many companies offer fine beads or granular particles of hypercrosslinked sorbents for SPE. Their main characteristics are presented in Table 9.7. 

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