Microfluidic device and sample analysis method

1 . A sample analysis method, comprising:

A) providing a microfluidic device, comprising:

a substrate having a microwell array comprising a plurality of microwells;

a peripheral member positioned on the substrate and configured to surround the microwell array of the substrate; and

a cover member comprising a surface formed integrally with the peripheral member such that the surface of the cover member faces the microwell array of the substrate, the surface of the cover member having an arithmetic average roughness, Ra, of 35 nm or less, a surface roughness, Rz, of 350 nm or less, and an Ra/Rz ratio of from 0.10 to 0.24;

wherein the cover member, the peripheral member, and the microwell array define a flow path configured to transport an aqueous sample to the plurality of microwells; and

wherein the aqueous sample contacts the surface of the cover member;

B) introducing an aqueous sample comprising a target molecule and a detection reagent into the flow path of the microfluidic device;

C) filling the plurality of microwells with the aqueous sample;

D) reacting the target molecule with the detection reagent to generate a signal for detection; and

E) detecting the signal to thereby characterize the sample and

wherein a signal originating from non-specific binding of the detection reagent to the surface of the cover member is either not detected or is detected from less than 10% of the detectable area of the surface of the cover member.

2 . The sample analysis method of claim 1 , wherein the detecting comprises determining with a false negative rate of less than 10% the number of the plurality of microwells having a detected signal.

3 . The sample analysis method of claim 1 , further comprising:

introducing a sealant into the flow path such that the sealant replaces the aqueous sample in the flow path and encapsulates the aqueous sample in the plurality of microwells of the microwell array in the substrate.

4 . The sample analysis method of claim 3 , further comprising:

heating the microfluidic device to promote the reacting.

5 . The sample analysis method of claim 1 , wherein the target molecule is selected from a DNA, an RNA, a protein, or a lipid.

6 . The sample analysis method of claim 1 , wherein the aqueous sample comprises cells.

7 . A sample analysis method, comprising:

A) providing a microfluidic device, comprising:

a substrate having a microwell array comprising a plurality of microwells;

a peripheral member positioned on the substrate and configured to surround the microwell array of the substrate; and

a cover member, comprising a mirror-polished surface integrally molded to the peripheral member such that the surface of the cover member faces the microwell array of the substrate, the surface of the cover member having an arithmetic average roughness, Ra, of 35 nm or less, a surface roughness, Rz, of 350 nm or less, and an Ra/Rz ratio of from 0.10 to 0.24;

wherein the cover member, the peripheral member, and the microwell array define a flow path configured to transport an aqueous sample to the plurality of microwells, the flow path having a first end connected to an injection port and a second end connected to a discharge port, the ports positioned inside the peripheral member and along the longitudinal direction of the cover member:

wherein the aqueous sample contacts the surface of the cover member; and

wherein the surface of the cover member is formed such that the surface of the cover member has a water contact angle of 85 degrees or more;

B) flushing the flow path of the microfluidic device with buffer;

C) introducing an aqueous sample comprising a target molecule and a detection reagent into the flow path such that the aqueous sample replaces the buffer in the flow path;

D) filling the plurality of microwells with the aqueous sample;

E) introducing a sealant into the flow path such that the sealant replaces the aqueous sample in the flow path and encapsulates the aqueous sample in the plurality of microwells of the microwell array in the substrate;

F) reacting the target molecule with the detection reagent to generate a signal for detection; and

G) detecting the signal to thereby characterize the sample;

wherein a signal originating from non-specific binding of the detection reagent to the surface of the cover member is either not detected or is detected from less than 10% of the detectable area of the surface of the cover member.

8 . The sample analysis method of claim 7 , wherein the detecting comprises determining with a false negative rate of less than 10% the number of the plurality of microwells having a detected signal.

9 . The sample analysis method of claim 7 , further comprising:

heating the microfluidic device to promote the reacting.