A bead-based immunogold-silver staining assay on capillary-driven microfluidics.

Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in "bead lanes", which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using "classical" enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL-1 of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL-1 of the same antibodies using a ~140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.

Visual detection of trace lead ion based on aptamer and silver staining nano-metal composite.

In this paper, visual detection of trace lead ion was established by aptamer and silver staining. The basic strategy was that aminated PS2.M aptamer was immobilized onto slide and formed stable G-quadruplex structure. PbS was generated by adding S2-, and it catalyzed subsequent silver staining reaction, through the silver staining amplification effect, the slide presented visible ash black. The gray value of slide after silver staining was analyzed and the semi-quantitative detection of Pb2+ in solution was realized. The results showed that optical darkness ratio (ODR) and logarithmic value of Pb2+ concentration had a good linear relationship (R2 = 0.951) over the range of 0.5-10 µM. In addition, there was no obvious interference of other common metal ions for the detection, indicating that this method presented outstanding selectivity. And it was also used for qualitative and semi-quantitative determination of Pb2+ in soil sample successfully.

Silver staining of SDS-polyacrylamide gel.

To detect nanogram quantities of protein and nucleic acids on SDS-PAGE gels.

Simple and cost effective apparatus for silver staining of polyacrylamide gels with sequential reagents addition and real time monitoring.

Highly reproducible results in molecular biology depend a lot on effective staining and destaining methods. Silver staining of polyacrylamide DNA and protein gel has been adopted widely in the molecular biology laboratories for detecting a very low nanogram range of sample. An efficient staining of a polyacrylamide gel requires a number of well controlled and highly sensitive steps that often becomes tiresome when done manually or when there are a number of gels to be stained simultaneously. Since, silver staining is a multistep procedure that requires proper fixation and exchange of substance, a reliable protocol is necessary and a simple apparatus may be an added advantage to carry out the steps with ease and safety. Here, we describe a simple and cost effective device made from off-the-shelf components for some established silver staining protocols. Staining is done on a tray while six graduated bottles with a liquid delivery stopcock each, is connected to the tray through silicon tubing. The used up solution is drained off completely from the staining tray through a liquid outlet stopcock using vacuum pressure. The system is fixed with a camera connected to a computer for effective control of the staining process in each step. The apparatus provides the researchers with efficient staining and real time monitoring of gels without the need for handling toxic chemicals.

Immunogold-silver staining-on-a-chip biosensor based on cross-flow chromatography.

Immunogold-silver staining (IGSS) was adopted in cross-flow chromatographic analysis in which immunological reactions and silver intensification were sequentially conducted in the vertical and horizontal directions, respectively. Factors controlling the performance, except the silver substrate solution, were optimized to increase the signal-to-background ratio in measurements of cardiac troponin I as a model analyte. In generating the signal, the size of colloidal gold catalyst was critical; the smallest size (5-nm diameter) in the selected range yielded the highest colorimetric signal. To maintain the low background, two processes, blocking the remaining surfaces of membrane after antibody immobilization and washing the residual tracer after immunological reaction, were necessary. Self-nucleation of silver ions also caused a background signal and was controlled to some degree by decreasing the hydrodynamic force that arose when the substrate solution was supplied in the horizontal direction. Finally, a new chip (IGSS-on-a-chip; IOC) that allowed for convenient, efficient IGSS was produced by injection molding of plastic. This method enhanced the detection capability by 51-fold compared to the conventional rapid test kit using 30nm-sized colloidal gold as the tracer. The IOC biosensor results also showed that silver intensification yield via cross flow after immunological reaction was 19% higher than that by traditional incubation.

Silver stains demonstrating neuroendocrine cells.

During the preimmunohistochemical era, silver stains were an important part of the staining arsenal for identifying certain tissue structures and cell types in tissue sections. Some of them were useful for demonstrating endocrine cells, especially in the gastrointestinal tract. Until the late 1950s, silver stains, particularly those identifying endocrine cells, were accompanied by a number of technical difficulties resulting from uncontrolled staining factors. In the 1960s, new silver stains were developed for endocrine cell types and these stains gave reproducible results. One of the "older" silver stains and two of the "newer" ones are emphasized in this presentation, namely the Masson, the Grimelius and the Sevier-Munger techniques. The Masson stain demonstrates the enterochromaffin (EC, serotonin) cells, the Grimelius stain is a broad endocrine cell marker, and the Sevier-Munger technique demonstrates EC and EC-like cells and the C-cells of the thyroid. Especially in the preimmunohistochemical era, these staining methods often were used for histopathological diagnosis, particularly the Grimelius technique. The silver stains were developed empirically, and with few exceptions the chemical background is not known. Staining protocols are included.

Bismuth tracing in organotypic cultures of rat hippocampus.

Bismuth is known to have neurotoxic side effects in humans and animals. In the 1970s France experienced about a thousand cases of patients suffering from bismuth-induced encephalopathy. Studies suggest that bismuth may provoke a selective degeneration of CA1 pyramidal cells in the organotypic cultures of rat hippocampus. A currently established technique for the histochemical visualization of bismuth was applied on hippocampal tissue cultures allowing the tracing of bismuth in concentrations hitherto not possible. The accumulation and subcellular localization of bismuth is demonstrated in the tissue cultures of rat hippocampus. CA1 pyramidal cells in the rat hippocampus exhibit the highest uptake of bismuth. High bismuth citrate concentrations (10 microM) are able to totally destroy the cytoarchitecture of the hippocampus. At ultrastructural levels bismuth was found to be located exclusively in lysosome-like organelles.

Rapid detection of fungal filaments in corneal scrapings by microwave heating-assisted Grocott's methenamine silver staining.

The Gomoris methanamine silver impregnation technique is a highly reliable and archiveable method of detecting fungal filaments, but the staining procedure is time consuming and laborious. A technique using microwave energy to reduce the duration of Gomori's silver staining is described.

A comparision between rapid Golgi and Golgi-Cox impregnation methods for 3-D reconstruction of neurons at the confocal scanning laser microscope.

We utilized two widely used impregnation methods, the silver "rapid Golgi" and the mercuric Golgi-Cox methods, for three-dimensional (3-D) reconstruction of neurons at the confocal scanning laser microscope (CSLM), to determine which of them was more suitable for this application. The Golgi-Cox method is the most consistent arid the cleanest procedure with respect to the "rapid Golgi" one which always produces samples with scattered reflective granules that interfere with the image formation at the CSLM. The interneuronal tissue in the case of Golgi-Cox impregnated specimens (i.e. the non-impregnated tissue among impregnated neurons) contributes less to the decrease of reflected light during z-sectioning than in the case of "rapid Golgi" impregnation, but the mercury impregnated samples reflect less than the silver impregnated ones. Owing to the necessity during deep z-scanning to adjust the sensitivity of the CLSM detector the acquisition of images from the deeper planes of the sample may be difficult. In our opinion the "sandwich" mounting of the specimen between two coverslips is indispensable in order to make it possible to scan it from both sides and, thus reduce the penetration in the sample and the consequent distortion of the image. Neither of the impregnation methods used is completely suitable for CLSM observations due both to their intrinsic limitations and to those imposed by the sample thickness.

Kinetic silver staining and quantification of proteins adsorbed to microtiter plates.

A silver stain was used to detect and quantitate proteins adsorbed to microtiter plate wells. The kinetics of the development of the silver stain were analyzed with an automated microtiter plate reader. The lag time for stain development was found to be a consistent indicator of the amount of protein adsorbed to a microtiter plate well. Protein which was not preadsorbed to the microtiter plate was not effectively stained by silver. Complete adsorption of protein applied to the microtiter plate was possible by drying small amounts of protein in very dilute buffers. Variations in sensitivity for different proteins were less than 30% for the panel of proteins examined. Determinations from kinetic silver staining agreed with those from copper staining for bovine albumin adsorbed to microtiter plates. The precision of kinetic silver staining assay was optimal in the range of 40 to 200 ng per microtiter plate well. In this range, the standard deviations averaged less than 5%. Even smaller amounts of protein can be detected and interpolated down to approximately 10 ng per well. The kinetic silver staining method can be used on standard microtiter plate readers without special filters and is readily adaptable to automated systems.