Bacteria detection using a colour silicon patch
The idea consists in the preparation of a colored biosensor made of a soft silicon patch coated with colored nanoscale particles made of polystyrene. The silicon patch can be glued or netted to any surface and or material.
The silicon patch will contain a covalently linked single stranded synthetic DNA (ssDNA) sequence; complementary to another DNA sequence attached to the surface of a colored (blue or red) nanoparticle polyester beads. Because of the complementarity between DNA bases (A-T; C-G), when the beads are deposited on the silicon surface, the hybridization reaction between both DNA sequences will generate a double stranded DNA (dsDNA) sequence containing regions with multiple sites for nucleases action. The hybridization between both DNA will provide a strong bound between the silicon and the polyester bead, generating a colored patch susceptible to the attack of bacteria and virus nucleases. The fact that the DNA strands are attached covalently to the surfaces of interest should result in a robust assembly method relatively insensitive to pH and temperature changes that would denature proteins.
See enclosed image containing a sketch of the proposed method. (At the end of the text)
Nucleases are a type of enzyme present in bacteria and virus that can hydrolyze double stranded DNA, breaking down the molecule at specific locations. They are known as biological scissor. A restriction endonuclease functions by "scanning" the length of a DNA molecule. Once it encounters its particular specific recognition sequence, it will bind to the DNA molecule and makes one cut in each of the two sugar-phosphate backbones. The positions of these two cuts, both in relation to each other, and to the recognition sequence itself, are determined by the identity of the restriction endonuclease used to cleave the molecule in the first place. Different endonucleases yield different sets of cuts, but one endonuclease will always cut a particular base sequence the same way, no matter what DNA molecule it is acting on. Once the cuts have been made, the DNA molecule will break into fragments.
Bacteria use this type of enzyme to protect themselves from certain types of virus, while virus uses these enzymes to introduce a gap in the host DNA molecule and as part of its replication machinery.
The proposed method only addresses the solution regarding bacteria identification. However, the method can be used for virus identification as long as viral nucleases target sequences are known.
Materials and methods
To prepare the silicon patch will be required: silicon mold, polystyrene beads and synthetic DNA uracil modified. The following sections describe the way in which these three components must be prepared according to Hartmann et al. (2002) with modifications. DNA sequences will be designed to allow the identification of bacterial and viral nucleases separately.
Preparation of DNA molecule for substrate attachment for bacteria identification
DNA molecules for substrate attachment are 31 bases pare in length, with two spacers made of triethylene glycols and a RNA uracil-nucleotide modification at their 3’ ends. (The sequences proposed in this document are completely different for those used in the Hartmann paper, and constitute an innovative application of this technology)
Substrate attached sequence
5 ‘ -GCGCGCGCGCCAAGCTTGAATTCCGCGCGCG– (2x triethylene glycols) – U-3’
MW- 9489 Da
Length – 31 bp
GC content – 74%
Melting temperature – 74°C (Note the high melting temperature above 70°C)
The sequence AAGCTTGAATTC provides recognition sites for two endonucleases EcoRI and HindIII from E coli and Haemophilus influenza respectively. The sequences can be modified depending on the microorganism that is going to be detected.
This sequence can be requested to any oligonucleotides supplier, and purchased lyophilized. The sequences must be reconstituted in 0.1M of EDTA buffer. Following reconstitution 30 optical units of DNA are mixed with 30 uL of 0.1M NaAcetate and 30uL of 0.45M NaIO4, and stirring for 2 h to achieve the complete oxidation of the ribose sugar of the uracil nucleotide at the 3’ end of each strand. Oxidized DNA must be desalted with G10 Sephadex column previously equilibrated with nanopure H2O. Fractions are collected and concentrated with butanol extraction. The concentrate is suspended in sodium phosphate (NaH2PO4, pH7.3) to produce a 0.1M of NaH2PO4 DNA solution.
Polystyrene bead attached sequence
5’- CGC GCG CGG AAT TCA AGC TTG GCG CGC GCG C - (2x triethylene glycols) – U-3’
This sequence is complementary to the substrate attached sequence, and has similar physical and chemical properties. The sequence has to be modified with the spacers (polyethylene glycols) and an aldehyde moiety following oxidation of the ribose sugar as described above. The modified DNA must be reconstituted in 0.45M NaAcetate.
Preparation of beads
Amine-functionalized polystyrene beads of 0.87 um are exchanged into 0.1M NaPhosphate, 0.15 M NaCl, pH7.2. by repeated buffer exchange and centrifugation. The beads are treated with a solution of succinimidyl 6- hydrazinonicotinamide acetone hydrazine (SANH) in N2N’ dimethylformamide and incubated at room temperature overnight. The beads are then centrifuged and the supernatant discarded. The beads are washed with 0.1 morpholinoethanesulfonic acid (MES), 0.9% NaCl, pH4.7 to exchange buffer and remove the hydrazone protecting group. Beads are then suspended in 0.45M NaAcetate.
Aldehyde functionalized DNA is mixed with the beads approximately at a ratio 1:10, and allowed to conjugate overnight. Beads are aspirated several times to remove unbound DNA and suspended in 0.1M NaCl, 50mM Tris, 0.1% Tween.
Preparation of substrates
Silicon mold substrates are placed for 5 min in a mixture of 10:1 toluene:aminopropylethoxysilane (APS) and heated to approximately 65°C. Substrate are then rinsed in toluene and methanol, dried with N2, and baked at 90°C for 1h.
The oxidized DNA is pipette onto the silicon surface, covered and allowed to stand 1 h at room temperature. The mold can be divided in two regions, one for bacteria contamination detection and the other for virus detection.
The mold is then rinsed vigorously in a 0.1 M solution of sodium phosphate, pH7.3 , 0.1% w/v sodium dodecyl sulfate (SDS) to remove non covalently attached DNA.
Bead substrate coupling
Coated beads are released in a droplet (~ 60uL) of 0.1 M of sodium phosphate (pH 7.3) solution above the substrate coated with the DNA fragment. Hybridization are allowed to occur for at least 24 h.
Problem this idea/invention addresses:
Bacteria are ubiquitous in the environment. They can contaminate any surface and are a major health problem. Detection requires specific laboratory methods. with this idea bacteria presence can be detected by simple visual inspection of a colored silicon patch.
Attached files:bacteria and virus detection 2.pdf Asking price:
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