Silver stain removal by chemical etching and sonication

Details Silver stain removal by chemical etching and sonication Silver stain removal by chemical etching and sonication

2001-11-30

2004-04-27

Alanko, Anita (Department: 1765)

Etching a substrate: processes

Nongaseous phase etching of substrate

Relative movement between the substrate and a confined pool...

C216S091000, C216S092000, C216S100000, C134S001000

Reexamination Certificate

active

06726847

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to method for regenerating spent DNA detection chips for further use. More specifically, the present invention relates to a method for the removal of silver from used DNA detection chips that employ gold nanoparticle-oligonucleotide conjugate probes and that use silver staining for signal amplification.
BACKGROUND OF THE INVENTION
Sequence-selective DNA detection has become increasingly important as scientists unravel the genetic basis of disease and use this new information to improve medical diagnosis and treatment. DNA hybridization tests on oligonucleotide-modified substrates are commonly used to detect the presence of specific DNA sequences in solution. The developing promise of combinatorial DNA arrays for probing genetic information illustrates the importance of these heterogeneous sequence assays to future science. In most assays, the hybridization of fluorophore-labeled targets to surface bound probes is monitored by fluoresecence microscopy or densitometry. Although fluoresence detection is very sensitive, its use is limited by the expense of the experimental equipment and by background emissions from most common substrates. In addition, the selectivity of labeled oligonucleotide targets for perfectly complementary probes over those with single base mismatches is poor, preventing the use of surface hybridization tests for detection of single nucleotide polymorphisms. A detection scheme which improved upon the simplicity, sensitivity and selectivity of fluorescent methods could allow the full potential of combinatorial sequence analysis to be realized.
One such technique is the chip based DNA detection method that employs gold nanoparticle probes, modified with oligonucleotides, to indicate the presence of a particular DNA sequence hybridized on a transparent substrate in a three component sandwich assay format. See T. A. Taton, C. A. Mirkin, R. L. Letsinger,
Science,
289, 1757 (2000). For low target DNA concentrations, the method employs nanoparticle-promoted silver reduction for signal amplification. The amplification step increases the sensitivity of the chip and provides for facile DNA detection. However, the silver cannot be readily removed from the chip by simply washing with water to reuse the chip as one can do with conventional fluorescence-based arrays. Accordingly, a method and composition for regenerating silver stained DNA nanoparticle-based chips for reuse is desirable.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for the removal of the silver from used DNA detection chips that employ silver staining for signal amplification. The invention relates to chemical compositions, methods using the chemical compositions and sonication methods for removing the silver.
Typically, a plurality of nanoparticle-oligonucleotide conjugates or oligonucletides can be attached to the substrate in an array for detecting multiple portions of a target nucleic acid, for detecting multiple different nucleic acids, or both. For instance, a substrate may be provided with rows of spots, each spot containing a different type of oligonucleotide or oligonucleotide-nanoparticle conjugate designed to bind to a portion of a target nucleic acid. A sample containing one or more nucleic acids is applied to each spot, and the rest of the assay is performed in one of the ways described above using appropriate oligonucleotide-nanoparticle conjugates, oligonucleotide-liposome conjugates, aggregate probes, core probes, and binding oligonucleotides such as the ones described in WO 98/04740, published Feb. 5, 1998 and WO 00/33079, published Jun. 8, 2000.
When a substrate is employed, a detectable change can be produced or further enhanced by silver staining. Silver staining can be employed with any type of nanoparticles that catalyze the reduction of silver. Preferred are nanoparticles made of noble metals (e.g., gold and silver). See Bassell, et al.,
J. Cell. Biol.,
126, 863-876 (1994); Braun-Howland et al.,
Biotechniques,
13, 928-931 (1992). If the nanoparticles being employed for the detection of a nucleic acid do not catalyze the reduction of silver, then silver ions can be complexed to the nucleic acid to catalyze the reduction. See Braun et al.,
Nature,
391, 775 (1998). Also, silver stains are known which can react with the phosphate groups on nucleic acids.
Silver staining can be used to produce or enhance a detectable change in any assay performed on a substrate. In particular, silver staining has been found to provide a huge increase in sensitivity for assays employing a single type of nanoparticle, such as the one illustrated in
FIG. 1
, so that the use of layers of nanoparticles, aggregate probes and core probes can often be eliminated.
In assays for detecting nucleic acids performed on a substrate, the detectable change can be observed with an optical scanner. Suitable scanners include those used to scan documents into a computer which are capable of operating in the reflective mode (e.g., a flatbed scanner), other devices capable of performing this function or which utilize the same types of optics, any type of grayscale-sensitive measurement device, and standard scanners which have been modified to scan substrates according to the invention (e.g., a flatbed scanner modified to include a holder for the substrate). The resolution of the scanner must be sufficient so that the reaction area on the substrate is larger than a single pixel of the scanner. The scanner can be used with any substrate, provided that the detectable change produced by the assay can be observed against the substrate (e.g., a gray spot, such as that produced by silver staining, can be observed against a white background, but cannot be observed against a gray background). The scanner can be a black and white scanner or a color scanner.
A problem associated with the silver enhancement technique is that the silver cannot be simply removed from the combinatorial DNA array substrates or chips that use oligonucleotide-modified gold nanoparticle probes and that employ nanoparticle-promoted silver reduction for signal amplication.
Accordingly, one object of the invention is to provide compositions for the removal of the silver from DNA detection chip, thereby allowing the chip to be recycled and reused.
Another object of the invention is to provide a cyanide-based method for removing silver from the used DNA detection chip allowing for the recycle and reuse of the chip.
Still another object of the invention is to provide a sonication method for the removal of silver and gold nanoparticles from a used DNA detection chip, thus allowing the chip to be recycled and reused.


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Bassell, et al., Single mRNA's Visualized by Ultrastructural in Situ Hybridization A

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