TWI761196B - Microfluidic biosensing chip integrating separate electrochemical electrodes - Google Patents

Abstract

A microfluidic chip includes: a base layer composed of impermeable material; a first electrode layer, including: a first substrate disposed on the base layer; a first electrode chip disposed on the first substrate and including at least one working electrode; a microfluidic channel layer made of impermeable material and having a microfluidic channel; a second electrode layer, including: a second electrode chip disposed on the microfluidic channel layer, and includes at least one reference electrode and at least one counter electrode; and a second substrate disposed on the second electrode chip; and a cover layer made of impermeable material, and disposed on the second electrode layer.

Description

Microfluidic detection chip integrating separate electrochemical electrodes

The present invention relates to a microfluidic chip, especially a microfluidic chip that integrates a reference electrode and an auxiliary electrode on the same chip, and sets a working electrode immobilized with biorecognition molecules on another chip and integrates it in a microfluidic channel wafer.

Affinity microfluidic chips can be used to detect chemical substances (such as proteins, biomarkers, nucleic acids and other biomolecules) in biological samples (such as blood, urine, saliva), and have a wide range of applications in the field of biomedicine value. How to improve the affinity reaction between the substance in the liquid in the microfluid and the biorecognition molecule on the sensing electrode (for example: antibody and antigen immunoreaction, nucleic acid probe and target nucleic acid fragment hybrid reaction, etc.) The manufacturing cost of the fluid wafer is an urgent problem to be solved in the related technical field.

TW 201016591 A discloses a biosensor package structure with microfluidic channels, which includes: a substrate having a first surface, a second surface and an opening; a biochip disposed on the first surface , and has a sensing area exposed on the opening; and an upper cover, disposed on the second surface, and covering the opening to form a micro-channel.

US 20110233059 A1 discloses a microfluidic sensor comprising a substrate sensor (1) and a structured polymer film (2).

WO 2014/144660 A1 discloses a point-of-care sensor system comprising a portable reader and a disposable kit for receiving and analyzing samples.

However, the microfluidic devices disclosed in the above-mentioned patent documents still have room for improvement in terms of detection performance and production cost.

In order to improve the detection performance and production cost of the prior art microfluidic device, the present invention provides a novel microfluidic chip.

In order to achieve the above-mentioned purpose and other purposes, the present invention provides a microfluidic chip, comprising: a base layer, which is composed of water-impermeable material; a first electrode layer, comprising: a first substrate, which is disposed on the base layer; a first electrode wafer, which is disposed on the first substrate and includes at least one working electrode; Wherein, the first base material has a first concave portion for accommodating the first electrode chip; a microfluidic channel layer, which is disposed on the first electrode layer, is made of water-impermeable material, and has a microfluidic channel; a second electrode layer, comprising: a second electrode wafer disposed on the microfluidic channel layer and comprising at least one reference electrode and at least one auxiliary electrode; and a second substrate disposed on the second electrode wafer; Wherein, the second substrate has a second recess for accommodating the second electrode chip; and a covering layer, which is made of water-impermeable material, is arranged on the second electrode layer; Wherein, the microfluidic wafer has two holes extending from the cover layer to two ends of the microfluidic channel, so that the microfluidic channel is in fluid communication with the outside world.

The above-mentioned microfluidic wafer, wherein the second electrode wafer may comprise: a center disk electrode, an inner ring electrode, which surrounds the center disk electrode, and an outer ring electrode, which surrounds the inner ring electrode, and the center The disk electrode, the inner ring electrode and the outer ring electrode are arranged concentrically, but the invention is not limited thereto. In another embodiment, the second electrode wafer may include a plurality of point electrodes, or electrodes of other types. .

In the above-mentioned microfluidic chip, the second substrate can be made of polydimethylsiloxane.

The above-mentioned microfluidic chip, wherein the first substrate may comprise: a transparent film; and Two glass slides are disposed on the transparent film, and the thickness of the two glass slides is substantially the same as the thickness of the first electrode chip, and the two glass slides are spaced apart from each other, so as to define the first glass slide between the two glass slides The concave portion is used to place the first electrode wafer, and there is substantially no height difference between the two glass slides and the first electrode wafer, so that the microfluidic channel layer can be flatly attached to the first electrode layer.

In the above-mentioned microfluidic chip, the two glass slides of the first substrate, the transparent film and the first electrode chip can be connected by a waterproof glue, so as to avoid leakage of the liquid in the microfluidic channel layer.

In the above-mentioned microfluidic wafer, the base layer, the microfluidic channel layer and the cover layer can be made of acrylic.

In the above-mentioned microfluidic chip, the base layer and the cover layer may have a plurality of holes corresponding to each other, and the holes are suitable for inserting a fastener to fix the base layer and the cover layer.

In the above-mentioned microfluidic wafer, the upper and lower surfaces of the microfluidic channel layer can be adhered with double-sided adhesive tape, which is beneficial for adhering the microfluidic channel layer to the first electrode layer and the second electrode layer, wherein the length of the microfluidic channel can be 39 mm mm, width can be 2 mm, height can be 1 mm.

The microfluidic wafer of the present invention integrates a reference electrode (RE) and a counter electrode (CE) on the same wafer, and disposes a working electrode (WE) on another wafer, It can avoid the contamination of the reference electrode and the auxiliary electrode when the affinity sensor is modifying the working electrode. At this time, the working electrode is more beneficial to become a multi-working electrode, which can be used for the immobilization of different biorecognition molecules and the detection of different targets. After electrode contamination or measurement, it is only necessary to replace the working electrode to continue to perform other affinity reactions, without the need to replace the reference electrode and auxiliary electrode. In addition, the first electrode layer and the second electrode layer of the microfluidic chip of the present invention respectively include a first substrate and a second substrate having a first recess and a second recess to accommodate the first electrode chip and the second substrate. The electrode chip can reduce the area covered by the first electrode chip and the second electrode chip in the overall micro-channel, so as to facilitate the mass production of sensing electrodes and reduce the electrode manufacturing cost.

The following describes the implementation of the present invention through specific embodiments, and those skilled in the art can understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and various details in this specification can also be modified and changed based on different viewpoints and applications without departing from the spirit of the present invention.

As used in the specification and the appended claims, the singular forms "a" and "the" include plural referents unless the context dictates otherwise.

As used in the specification and the appended claims, the term "or" includes the meaning of "and/or" unless the context otherwise requires.

Example 1

As shown in FIG. 1 and FIG. 2 , the microfluidic wafer 1 according to Embodiment 1 of the present invention includes: a base layer 10 , which is made of a water-impermeable material; a first electrode layer 11 , including: a first substrate 111, which is arranged on the base layer 10; a first electrode wafer 112, which is arranged on the first substrate 111 and includes at least one working electrode 113; wherein, the first substrate has a first recess 114 for accommodating the first electrode wafer 112; a microfluidic channel layer 12 disposed on the first electrode layer 11 and made of water-impermeable material and having a microfluidic channel 121; a second electrode layer 13, comprising: a second electrode wafer 131, which is disposed on the microfluidic channel layer 121 and includes at least one reference electrode 132 and at least one auxiliary electrode 133; and a second substrate A material 134 is disposed on the second electrode chip 131; wherein, the second substrate 134 has a second recess 135 for accommodating the second electrode chip 131; and a cover layer 14, which is It is made of water-impermeable material and is disposed on the second electrode layer 13; wherein, the microfluidic wafer 1 has two holes 15 extending from the cover layer 14 to the two ends of the microfluidic channel 121, so that the microfluidic chip 1 has two holes 15. The fluid channel 121 is in fluid communication with the outside world.

In use, a liquid to be side is injected into the microfluidic channel 121 of the microfluidic chip 1 from a hole 15, so that the liquid to be sided flows along the microfluidic channel 121 and flows out from another hole 15, So that the liquid to be side flows through the working electrode 113 , the reference electrode 132 and the auxiliary electrode 133 , so as to detect chemical substances (such as proteins, biomarkers, nucleic acids and other biomolecules) contained in the liquid to be side.

In a preferred embodiment of the present invention, the second substrate is made of polydimethylsiloxane, but the present invention is not limited to this, and those with ordinary knowledge in the technical field of the present invention can follow the actual needs Choose other suitable materials.

In a preferred embodiment of the present invention, the first substrate comprises: a transparent film; and two glass slides, which are disposed on the transparent film, and the two glass slides are spaced apart from each other, so that the glass slides The first concave portion is defined between wafers, but the present invention is not limited thereto, as long as the first concave portion can be appropriately formed to accommodate the first electrode wafer.

In a preferred embodiment of the present invention, the first substrate and the first electrode chip are connected by a waterproof glue, but the present invention is not limited to this, as long as the first substrate and the first electrode can be properly fixed The relative displacement of the first electrode wafer can be prevented.

In a preferred embodiment of the present invention, the base layer, the microfluidic channel layer and the cover layer are made of acrylic, but the present invention is not limited to this, those skilled in the art to which the present invention pertains are Other suitable materials can be selected according to actual needs.

In a preferred embodiment of the present invention, the base layer and the cover layer have a plurality of holes corresponding to each other, and the holes are suitable for inserting a fastener to fix the base layer and the cover layer, but The present invention is not limited to this, as long as the layers of the microfluidic wafer can be properly fixed.

In a preferred embodiment of the present invention, the length of the microfluidic channel is 39 mm, the width is 2 mm, and the height is 1 mm, but the present invention is not limited to this. Knowledgeable people can choose the appropriate size according to actual needs.

In a preferred embodiment of the present invention, the surface of the working electrode is modified with antibody molecules, but the present invention is not limited to this, and the surface of the working electrode can also be unmodified or modified with other biomolecules.

Example 2

The microfluidic wafer of Example 2 is substantially the same as that of Example 1, and the difference is only in the structures of the first electrode wafer and the second electrode wafer.

As shown in FIG. 3 , the first electrode wafer 3 of the microfluidic wafer of Example 2 includes a first working electrode 31 , a second working electrode 32 and a third working electrode 33 , and the working electrodes are partially covered by an insulating layer 34 overlays.

Compared with Example 1, the microfluidic chip of Example 2 includes a plurality of working electrodes, which can further improve the multiplexing during detection.

As shown in FIG. 4 and FIG. 5 , the second electrode wafer 4 of the microfluidic wafer of Example 2 includes: a center disk electrode 41 , an inner ring electrode 42 surrounding the center disk electrode 41 , and an outer ring electrode 43, which surrounds the inner ring electrode 42, and the central disk electrode 41, the inner ring electrode 42 and the outer ring electrode 43 are arranged concentrically.

The center disk electrode 41 of the second electrode wafer 4 of the microfluidic wafer of Example 2 can be used as a reference electrode, and the inner ring electrode 42 and the outer ring electrode 43 can be used as auxiliary electrodes and spare auxiliary electrodes respectively, so as to further improve the stability of detection sex.

It should be understood that the dimensions of the components shown in FIG. 4 and FIG. 5 are only examples, but the present invention is not limited thereto, and those skilled in the art to which the present invention pertains can select appropriate dimensions according to actual needs.

Test Example 1

To confirm the detection performance of the microfluidic chip of the present invention, the following tests were performed:

The microfluidic wafer of Example 2 was taken, the surface of the working electrode was plated with gold, and the surface of the working electrode was modified with Ara h 1 antibody.

A true sample of Ara h 1 at a concentration of 1127.5 µg/ml was obtained after the extraction step of standard peanut butter. The Ara h 1 real sample was diluted to 1 µg/ml with 10 mM Phosphate buffered solution (PBS), and the Ara h 1 was diluted with 10 mM PBS and 2.5 mM Fe(CN) ₆ ^3-/4- 1 The real sample was diluted to 100 pg/ml, and the liquid was exchanged three times in a fixed mode of liquid exchange in the microfluidic channel, and the change in _Ret was measured after the liquid exchange. The Nai diagram is shown in Figure 6.

In addition, using the standard sample of the same concentration (100 pg/ml) as a control, three times of liquid exchange were also performed in the microfluidic channel in a fixed mode of exchange, and the change of _Re was measured after the exchange. _ΔRet was plotted against the number of fluid changes, and the results are shown in FIG. 7 .

As can be seen from Figure 6 and Figure 7, taking the detection of Ara h 1 as an example, the detection values of the real sample and the standard sample are very similar, and the error rate is within 10%, and more Ara h can be provided after the liquid is changed. 1 molecule, increase the amount of immune bonding on the surface of the working electrode, so that ΔR _et increases with the increase of the number of liquid changes, which can prove that the microfluidic chip of the present invention has a good detection performance.

To sum up the above, the microfluidic chip of the present invention has at least the following excellent technical effects: 1. Affinity sensors generally immobilize biorecognition molecules (such as antibodies, probe DNA, and aptamers) on the surface of a working electrode (WE) to detect specific molecules. For example, a working electrode whose surface is modified by an antibody molecule can detect a specific antigen by the affinity between the antibody molecule and a specific antigen. Traditional microfluidic wafers integrate working electrode (WE), reference electrode (RE) and counter electrode (CE) on the same wafer, which may cause contamination at the same time when modifying the working electrode. The reference electrode and the auxiliary electrode, which in turn affect the detection performance. In contrast, in the microfluidic chip of the present invention, the reference electrode and the auxiliary electrode are integrated on the same wafer, and the working electrode is arranged on another wafer, so that the reference electrode and the auxiliary electrode can be avoided when the working electrode is modified. contamination, thereby improving its detection performance. 2. The first electrode layer and the second electrode layer of the microfluidic chip of the present invention respectively include a first substrate and a second substrate having a first recess and a second recess to accommodate the first electrode chip and the second substrate. The electrode chip can reduce the area occupied by the first electrode chip and the second electrode chip on the first substrate and the second substrate, so as to facilitate mass production of the electrode chip and reduce the cost of manufacturing and measurement.

The above-mentioned embodiments are only used to illustrate the present invention, but not to limit the present invention. Any person skilled in the art can modify and change the above embodiments without departing from the spirit and scope of the present invention. Therefore, the protection scope of the right of the present invention should be as set forth in the patent application scope described later.

1: Microfluidic chip 10: Basal layer 11: The first electrode layer 111: The first substrate 112: The first electrode wafer 113: Working electrode 114: The first recess 12: Microfluidic channel layer 121: Microfluidic Channels 13: Second electrode layer 131: Second electrode wafer 132: Reference electrode 133: auxiliary electrode 134: Second substrate 135: Second recess 14: Overlay 15: Holes 3: The first electrode wafer 31: The first working electrode 32: Second working electrode 33: The third working electrode 34: Insulation layer 4: The second electrode wafer 41: Center disk electrode 42: Inner ring electrode 43: Outer ring electrode

1 is a cross-sectional view of a microfluidic wafer according to Example 1 of the present invention. Fig. 2 is an exploded view of the microfluidic chip of Example 1 of the present invention. 3 is a schematic diagram of the first electrode wafer of the microfluidic wafer according to the second embodiment of the present invention. 4 is a schematic diagram of the second electrode wafer of the microfluidic wafer according to the second embodiment of the present invention. 5 is an enlarged schematic view of the second electrode wafer of the microfluidic wafer according to the second embodiment of the present invention. [ Fig. 6 ] is a Nye diagram of Test Example 1 of the present invention. [Fig. 7] is the result of plotting the number of fluid changes versus ΔR _et in Test Example 1 of the present invention.

1: Microfluidic chip

10: Basal layer

11: The first electrode layer

111: The first substrate

112: The first electrode wafer

113: Working electrode

114: The first recess

12: Microfluidic channel layer

121: Microfluidic Channels

13: Second electrode layer

131: Second electrode wafer

132: Reference electrode

133: auxiliary electrode

134: Second substrate

135: Second recess

14: Overlay

15: Holes

Claims (8)

A microfluidic chip, comprising: a base layer, which is composed of water-impermeable material; a first electrode layer, comprising: a first substrate, which is disposed on the base layer; a first electrode wafer, which is disposed on the first substrate and includes at least one working electrode; Wherein, the first base material has a first concave portion for accommodating the first electrode chip; a microfluidic channel layer, which is disposed on the first electrode layer, is made of water-impermeable material, and has a microfluidic channel; a second electrode layer, comprising: a second electrode wafer disposed on the microfluidic channel layer and comprising at least one reference electrode and at least one auxiliary electrode; and a second substrate disposed on the second electrode wafer; Wherein, the second substrate has a second recess for accommodating the second electrode chip; and a covering layer, which is made of water-impermeable material, is arranged on the second electrode layer; Wherein, the microfluidic wafer has two holes extending from the cover layer to two ends of the microfluidic channel, so that the microfluidic channel is in fluid communication with the outside world. The microfluidic wafer of claim 1, wherein the second electrode wafer comprises: a center disk electrode, an inner ring electrode surrounding the center disk electrode, and an outer ring electrode surrounding the inner ring electrode, and The central disk electrode, the inner ring electrode and the outer ring electrode are arranged concentrically. The microfluidic chip of claim 1, wherein the second substrate is made of polydimethylsiloxane. The microfluidic wafer of claim 1, wherein the first substrate comprises: a transparent film; and Two glass slides are disposed on the transparent film, and the thickness of the two glass slides is substantially the same as the thickness of the first electrode chip, and the two glass slides are spaced apart from each other, so as to define the first glass slide between the two glass slides The concave portion is used to place the first electrode wafer, and there is substantially no height difference between the two glass slides and the first electrode wafer, so that the microfluidic channel layer can be flatly attached to the first electrode layer. The microfluidic wafer of claim 1, wherein the two glass slides, the transparent film and the first electrode wafer of the first substrate are connected by a waterproof glue to avoid leakage of liquid in the microfluidic channel layer. The microfluidic wafer of claim 1, wherein the base layer, the microfluidic channel layer and the cover layer are made of acrylic. The microfluidic chip of claim 1, wherein the base layer and the cover layer have a plurality of holes corresponding to each other, and the holes are suitable for inserting a fastener to fix the base layer and the cover layer. The microfluidic wafer of claim 1, wherein the upper and lower surfaces of the microfluidic channel layer are adhered with double-sided adhesive tapes, which facilitates the adhesion of the microfluidic channel layer to the first electrode layer and the second electrode layer, wherein the length of the microfluidic channel is The system is 39 mm, the width is 2 mm, and the height is 1 mm.

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