Device and method for quantitatively determining an analyte, a method for determining an effective size of a molecule, a method for attaching molecules to a substrate, and a device for detecting molecules
a quantitative determination and analyte technology, applied in the field of quantitative determination devices and methods, can solve the problems of degradation of detection sensitivity, increased background noise, and difficulty in quantitative determination of analy
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example 1
[0161] This example relates to a DNA chip wherein a single-stranded DNA is installed on a detecting unit as a capturing unit, to detect the concentration of a complementary DNA or an analyte in an aqueous solution.
[0162] The outline of an exemplary DNA chip of this example and its basic principle of operation will be explained, using FIGS. 4-A to 4-C. First, as shown in FIG. 4-A, a Au thin film is installed as a detecting unit 13 at the bottom of part of a 1-mm wide flow channel in an elongated shape along the flow channel. A single-stranded DNA 42 having an SH group on one end as a capturing unit is attached onto the Au thin film 13 at a surface density of 5×1012 molecules / cm2 (see Analytical Chemistry, vol. 70, p. 4670-4677, 1998, for example), to make capturing units. A two-dimensional CCD light-receiving unit 41 is installed over the flow channel with the single-stranded DNA attached thereto, for observing fluorescent signals from the capturing units.
[0163] Next, as shown in F...
example 2
[0166] This example relates to determining the effective radius of a single-stranded oligonucleotide in an aqueous solution, and further, to controlling the attaching density of oligonucleotide attached to a substrate.
[0167] Synthesized was a single-stranded 24-mer oligonucleotide to which a thiol group having a C6H12 alkyl chain (—C6H12—SH) at the 3′ terminal was introduced, and the thiol group was reacted with a polished Au electrode at room temperature for 30 minutes, to bind the single-stranded oligonucleotide to the electrode. The thiol group may be introduced to an intermediate section of a single strand or at the 5′ terminal. The aqueous solution used for the reaction contained 10 mM of a Tris buffer (2-amino-2-hydroxymethylpropane-1,3-diol), and adjusted to have a pH of 7.4.
[0168] NaCl was used as an electrolyte. The screening effect of a negatively charged oligonucleotide was controlled, by changing the ion concentration of the electrolyte (salt concentration). Since the ...
example 3
[0173] As a related example, the above-described method was employed, and single-stranded, 12-mer oligonucleotide molecules having a fluorescent pigment at an end, and a thiol group at the other end were attached to a Au electrode. The result of monitoring the fluorescence from the pigment with varying the density of the attached molecules is shown in FIG. 12. This fluorescent pigment decreases or extinguishes the light by the quenching effect when it comes close to or contacts with the Au substrate, and emits or increases the light when it goes away from the substrate.
[0174] From FIG. 12, it is evident that by varying the attaching density, there appear three regions observed: (1) a low density region where adjacent oligonucleotide molecules do not interfere with each other, and the fluorescence intensity is dependent only on the increase in the attaching density; (2) a intermediate density region where steric hindrance between adjacent oligonucleotide molecules occurs, forcing th...
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