RESUMO
As a first step for the development of biosensing inks for inexpensive paper-based biodetection, we prepared paper strips printed with carboxylic poly( N-isopropylacrylamide) microgels that were modified either with an antibody or with a DNA aptamer. We found that the antibody and the DNA aptamer retained their recognition capabilities when coupled to microgel. The printed microgel remains stationary during chromatographic elution while the microgel-supported molecular recognition elements are accessible to their intended targets present in the elution solution. Our work indicates that microgels, large enough to isolate the biosensors from the paper surface, are sufficiently hydrophilic to be wetted during chromatographic elution, exposing the gel-supported affinity probes to their targets.
Assuntos
Acrilamidas/química , Técnicas Biossensoriais , Géis/química , Tinta , Papel , Polímeros/química , Resinas Acrílicas , Trifosfato de Adenosina/análise , Anticorpos/química , Antígenos/análise , Aptâmeros de Nucleotídeos/química , Cromatografia em PapelRESUMO
We demonstrate that DNA oligonucleotides covalently coupled to colloidal microgel can be manipulated by T4 DNA ligase for DNA ligation and by Phi29 DNA polymerase for rolling circle amplification (RCA). We also show that the long single-stranded RCA product can generate intensive fluorescence upon hybridization with complementary fluorescent DNA probe. We believe DNA-microgel conjugates can be explored for the development of DNA based bioassays and biosensors.
Assuntos
DNA Ligases/química , DNA Polimerase Dirigida por DNA/química , DNA/química , Géis/química , Sequência de Bases , Bioensaio/métodos , Técnicas Biossensoriais/métodos , DNA/metabolismo , DNA Ligases/metabolismo , Sondas de DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Dados de Sequência MolecularRESUMO
With the long-term goal of developing paper surfaces that will detect pathogens, we have investigated physical adsorption and covalent coupling as strategies for treating cellulose surfaces with a DNA aptamer that binds ATP. Physical adsorption was reversible and the isotherms fitted the Langmuir equation with an adsorption maximum of 0.105 mg/m2 at high ionic strength (300 mM NaCl, 25 mM Tris-HCl) and only 0.024 mg/m2 in lower ionic strength buffer (25 mM Tris-HCl). Covalent coupling of amine-terminated aptamer with oxidized cellulose film (Schiff base + reduction) gave 25% coupling efficiency while maintaining the aptamer activity which was illustrated by using a known fluorescent aptamer that is capable of ATP detection. Therefore, covalent coupling, without spacer molecules, is a promising approach for supporting biosensing aptamers on cellulose.
