HK1108008A - Electrochemical assay device and related methods - Google Patents

Description

Electrochemical assay device and related methods

This application claims the benefit of U.S. provisional application 60/521,555 filed on 21/5/2004, the entire contents of which are incorporated herein by reference.

Background

The present application relates to electrochemical analysis devices in the form of single use test strips for detecting the presence or quantity of an analyte residing in a sample and methods of making and using the same.

Single use disposable test strips for the electrochemical detection of an analyte such as glucose are known. In these test strips, a sample is introduced into the test strip to contact at least two electrodes. The oxidation or reduction of the analyte is observed as a current generated between the two electrodes. For example, using glucose detection in a conductivity cell (cell), as shown in fig. 1, glucose is oxidized by an enzyme (glucose oxidase) to form gluconolactone and a reduced enzyme. Regeneration of the oxidized form of the enzyme proceeds by reaction with the oxidized mediator, resulting in a reduced mediator. The reduced mediator transfers electrons to one electrode, while at the other electrode, electrons are transferred to the oxidized mediator, thus producing an observable current. FIG. 2 shows the observed current as a function of time in a test strip using an enzyme/mediator reagent system. In this figure, t-0 is the time of sample application. As shown, the current rises through a maximum and then falls to a final steady-state plateau. Measurements to determine the amount of analyte are taken after the maximum current is reached, and are typically taken after a steady state is reached.

In fig. 2, a delay is observed that may constitute a significant part of the total measurement time before the maximum value is reached. The duration of this delay depends on the distance between the electrodes and the fluidity of the medium used in the test strip. The medium fluidity is a property of the medium itself, i.e. the diffusion coefficient, but it also depends on other sample properties such as hematocrit and viscosity.

To make it more convenient for the user, improvements to analyte test strips in general, and glucose test strips in particular, have focused on two main goals: shorter test times and less sample volume. To some extent, these two goals have been achieved in tandem, since smaller sample volumes use smaller cells with smaller electrode-spacing, and smaller electrode-spacing results in shorter reaction times. However, these cells still have the current/time curve of fig. 2, and therefore a delay is embedded before the measurement can be made. The present invention eliminates this delay and therefore the time required to complete the test is significantly reduced.

Disclosure of Invention

According to the present invention, an electrochemical test device is provided having a base layer with a first electrode thereon and a top layer with a second electrode thereon. The two electrodes are separated by a spacer layer having an opening therein such that a space for receiving a sample is defined by one electrode on the top surface, the other electrode on the bottom surface, and a sidewall formed by edges of the opening in the spacer.

In a conductivity cell, where the reagents for performing the electrochemical reaction are deposited on one electrode instead of the side wall, the device produces a signal curve as shown in fig. 2. In the device of the invention, the reagent is deposited not only on the electrode, but also on the side walls of the space receiving the sample (fig. 3). This results in no or reduced hysteresis in the signal curve (fig. 4). Although only 25% of the sidewall height is coated with the agent to achieve a significant improvement, in a preferred embodiment the agent is spread out over the full height of the sidewall.

The present invention also provides a method of making the test strip of the present invention. According to the method, an intermediate structure is formed that includes a base layer and a spacer layer disposed on the base layer. The base layer has a first electrode disposed thereon, and the electrode is exposed through an opening in the spacer layer. Thus, the edges of the first electrode/base layer and the open spacer layer define a well or channel. The reagent-containing liquid is introduced into the well or trench such that it at least partially, and preferably completely, covers the side walls of the well. The liquid is then dried, leaving a coating of reagent on the bottom (first electrode) and side walls of the trough/trench. Thereafter, a top layer and a second electrode are added over the trench/groove.

In a preferred embodiment of the method, the spacer layer has an adhesive coating and a release sheet (release sheet) on the opposite side of the base layer, and the sidewalls of the channels extend upwardly through the release sheet. Reagent material is introduced into the grooves/channels such that at least some portion of the release sheet side walls are covered with reagent-containing liquid prior to drying, and preferably covered with reagent after drying. The release sheet is then removed to form a trough in which the sidewalls are substantially completely covered with the dried reagent.

Brief Description of Drawings

FIG. 1 shows the basic chemistry for a glucose test strip.

FIG. 2 shows the current as a function of time in a conductivity cell test strip with reagent applied only to the bottom of the well.

Fig. 3 shows a cross-section of the sample receiving space of the device according to the invention.

FIG. 4 shows current as a function of time in a test strip according to the present invention.

Fig. 5 shows a cross-section of the sample receiving space of the device according to the invention.

Figure 6 shows a schematic representation of the process of the invention.

FIGS. 7A and B show diagrammatic representations of the process of the present invention.

Detailed Description

The present application relates to electrochemical test devices or strips of the type commonly used for blood glucose analysis.

Definition of

As used in the specification and claims of this application, the term "electrochemical test device" refers to a device for determining an analyte in a sample using electrochemical analysis, either alone or in combination with a reusable meter. The preferred electrochemical test device is a disposable single use device of the type commonly known for home use in determining glucose levels.

The term "analyte" as used in the specification and claims of this application refers to a component of a sample to be measured. Non-limiting examples of specific analytes include glucose, hemoglobin, cholesterol, and vitamin C.

The term "electrode" as used in the specification and claims of this application refers to a component of an electrochemical test device that transfers electrons to or from a substance in a sample introduced into a sample-receiving space of the device, and which is connected or connectable to an electrical circuit as a current or potential difference between electrodes contacting the same sample to determine the amount of electron transfer present. The electrodes of the device of the present invention are made of a conductive material that is compatible with the particular analyte that the electrochemical cell is intended to detect. Specific examples of suitable conductive electrode materials include gold, carbon, silver, palladium, and platinum. The conductive materials for the first and second electrodes may be the same, or they may be different from each other. In a preferred embodiment of the invention, the conductive material used to form the electrodes is gold.

As used in the specification and claims of this application, the term "spacer" refers to a layer of material that provides electrical isolation between two electrodes of a device. Thus, the spacing is typically an insulating material and electrical contact between the electrodes only occurs if a sample is present in the sample-receiving space. In a preferred embodiment, the spacer is formed from a thin film or sheet of insulating material. Examples of suitable materials include, without limitation, polyimide, polyester, polyethylene terephthalate (PET), polycarbonate, glass, and fiberglass. The spacers may also be formed by deposition of an insulating layer, for example by spraying on a resistive coating. The openings may be formed in the above layers using conventional techniques including pre-cut openings in a defined film or sheet, laser or chemical etching, and the like.

As used in the specification and claims of this application, the term "reagent" refers to a chemical or mixture of chemicals that, when mixed with a sample, allows an electrochemical test device to be used to determine an analyte in the sample. The reagents need not be sufficient to make this determination and although not preferred, it is acceptable to add more chemicals to the sample prior to introduction to the test device. However, the reagent does at least comprise a redox active species which is oxidised at the first electrode and reduced at the second electrode (or vice versa) when the device is in use. The reagent may include a variety of redox active materials that act as charge carriers between the electrodes.

As used in the specification and claims of this application, the phrase "determining an analyte" refers to and encompasses qualitative detection of the presence of an analyte (i.e., whether the analyte is present in a detectable amount in a sample), semi-quantitative detection (i.e., whether the analyte is present in an amount greater than a predetermined threshold), and quantitative evaluation (i.e., determining the actual amount of analyte present).

As used in the specification and claims of this application, the term "covering" refers to coating a designated surface. Complete coverage is not required, for example in the case where pinholes may be present in the coating, but only the distribution of the covering agent over the designated surface. In addition, it is not excluded that the coating may be less than full area, due to unintended drawbacks of the coating method in a specific device.

As used in the specification and claims of this application, the phrase "sidewall portion extending continuously from a covered electrode" refers to a coating in which the reagent coating on the electrode on the sidewall merges into the reagent coating.

Apparatus of the invention

Fig. 3 shows a cross-section of the sample receiving space of the device according to the invention. As shown, the substrate layer 31 has an electrode 32 disposed thereon. The spacer layer 33 has an opening therein which provides sidewalls 34, 34'. The top substrate layer 35 has an electrode 36 disposed thereon. A sample receiving space 37 is bounded by the electrodes 32 and 36 and the side walls 34 and 34' and contains a dry reagent 38. In an alternative embodiment, the sample receiving space may have top and bottom surfaces, which are partly covered by the electrodes and partly expose the top layer. The dry reagent 38 covers the electrode 32 at the bottom of the sample receiving space 37 and extends upwardly along the side walls 34, 34'.

Fig. 4 shows a current/time curve for a device according to the invention, wherein the dry reagent covers substantially all of the side walls. This graph shows a clear advantage of the invention compared to fig. 2, i.e. that the current appears immediately and the steady state is achieved in a shorter time.

Without intending to be bound by any particular mechanism, it is believed that this effect occurs because charge carriers are present from the beginning at a location close to both electrodes, and thus can immediately generate current. Conversely, when a reagent is applied alone to the surface of the first electrode, the chemical reaction may begin immediately upon sample addition, but the true current cannot flow until the mediator (or some other redox active species) diffuses from the first electrode to the second electrode. This takes time and therefore there is a delay before the analyte-dependent current is observed. In addition, in the case of reagents with a small amount of active mediator, beyond a certain point the reaction cannot even start until the reverse reaction exists. This delay in the initiation of the chemical reaction is cumulative with the delay caused by other diffusion processes.

Based on this mechanism, it is theoretically expected that the delay in time will be related to the square of the distance that the charge carriers must travel to reach the second electrode. This means that if the distance between the dry reagent and the second electrode is reduced to 1/2, the time will be reduced to 1/4 and even coating 25% of the side wall will result in the time required to reach the current maximum being reduced to about 1/2. Thus, in the device of the invention at least 25%, preferably at least 50%, more preferably at least 75% and most preferably all of the side walls extending above the first electrode are coated with dry reagent.

This mechanism also clearly illustrates that an important component in the reagent is the mediator or charge carrier. Thus, as shown in fig. 5, in an alternative embodiment of the invention, a reagent layer 51 comprising an enzyme, such as glucose oxidase, is deposited on the surface of the first electrode, and a redox-active coating 52 is deposited to cover the bottom and at least part of the sides of the sample-receiving space.

The redox-active coating 52, or the reagent coating 38, may contain the redox state of the redox-active species used in the device. This may be in the reduced form, the oxidized form or a mixture thereof. Specific non-limiting examples of redox active materials are redox mediators known for use in glucose and other mediated electrochemical detection systems. The term "redox mediator" as used in the specification and claims of this application refers to a chemical species other than an analyte that is oxidized and/or reduced during a multi-step process such that electrons are transferred from the analyte to an electrode of an electrochemical cell or vice versa. Non-limiting examples of media include:

cyhaloferrite

[FeIII(CN)5(ImH)]^2-

[FeIII(CN)5(Im)]^3-

[RuIII(NH3)5(ImH)]³⁺

[RuIII(NH3)5(Im)]²⁺

[FeII(CN)5(ImH)]^3-

[RuII(NH3)5(Im)H]²⁺

[(NC)5FeII(Im)RuIII(NH3)5]^-

[(NC)5FeIII(Im)RuIII(NH3)5]⁰

[(NC)5FeII(Im)RuII(NH3)5]^2-

Ferrocene (Fc) and derivatives, including but not limited to:

ferrocene monosulfonate

Ferrocene disulfonate

FcCO₂H

FcCH2CO₂H

FcCH:CHCO₂H

Fc(CH₂)₃CO₂H

Fc(CH₂)₄CO₂H

FcCH₂CH(NH₂)CO₂H

FcCH₂SCH₂CH(NH₂)CO₂H

FcCH₂CONH₂

Fc(CH₂)₂CONH₂

Fc(CH₂)₃CONH₂

Fc(CH₂)₄CONH₂

FcOH

FcCH₂OH

Fc(CH₂)₂OH

FcCH(Me)OH

FcCH₂O(CH₂)₂OH

1，1′-Fc(CH₂OH)₂

1，2-Fc(CH₂OH)₂

FcNH₂

FcCH₂NH₂

Fc(CH₂)₂NH₂

Fc(CH₂)₃NH₂

1，1′-Me₂FcCH₂NH₂

FcCH₂NMe₂

(R)-FcCH(Me)NMe₂

(S)-FcCH(Me)NMe₂

1，2-Me₃SiFcCH₂NMe₂

FcCH₂NMe₃

FcCH₂NH(CH₂)₂NH₂

1，1′-Me₂FcCH(OH)CH₂NH₂

FcCH(OH)CH₂NH₂

FcCH:CHCH(OH)CH₂NH₂

Fc(CH₂)₂CH(OH)CH₂NH₂

FcCH₂CH(NH₂)CH₂OH

FcCH₂CH(CH₂NH₂)CH₂OH

FcCH₂NH(CH₂)₂OH

1，1′-Me₂FcCHOCONHCH₂

FcCH(OH)(CH₂)₂NH₂

1，1′-Me₂FcCH(OH)CH₂NHAc

FcB(OH)₃

FcC₆H₄OPO₃Na₂

Tris (phenanthroline) osmium II and osmium III (i.e., Os-phen) complexes, including but not limited to:

Os(4，7-dmphen)₃

Os(3，4，7，8-tmphen)₃

Os(5，6-dmphen)₃

Os(bpy)₃Cl₂

Os(5-mphen)₃

Os(5-Cl-phen)₃

Os(5-NO₂-phen)₃

Os(5-phphen)₃

Os(2，9-dm-4，7-dpphen)₃

and isomorphic ruthenium complexes, including but not limited to:

Ru(4，7-dmphen)₃

Ru(3，4，7，8-tmphen)₃

Ru(5-mphen)₃

Ru(5，6-dmphen)₃

Ru(phen)₃

[Ru(4，4′-diNH₂-bipy)3]²⁺

tris (bipyridine) osmium II and osmium III complexes (i.e., Os (bpy)₃) Including, but not limited to:

Os(bpy)₃

Os(dmbpy)₃

and related ruthenium complexes, such as:

Ru(bpy)₃

Ru(4，4’-diNH₂-bpy)₃

Ru(4，4’-diCO₂Etbpy)₃

bis (bipyridyl) osmium II and osmium III (i.e., Os (bpy)2) complexes with other ligands, including but not limited to:

Os(bpy)₂dmbpy

Os(bpy)₂(HIm)₂

Os(bpy)₂(2MeHIm)₂

Os(bpy)₂(4MeHIm)₂

Os(dmbpy)₂(HIm)₂

Os(bpy)₂Cl(HIm)

Os(bpy)₂Cl(1-MeIm)

Os(dmbpy)₂Cl(HIm)

Os(dmbpy)₂Cl(1-MeIm)

and related ruthenium complexes, such as:

Ru(bpy)₂(5，5’diNH₂-bpy)

Ru(bpy)₂(5，5’diCO₂Etbpy)

Ru(bpy)₂(4，4’diCO₂Etbpy)

wherein Et is ethyl, bpy is bipyridyl, dmbpy is dimethylbipyridyl, MeIm is N-methylimidazole, MeHIm is methylimidazole, HIm is imidazole, phen is phenanthroline, mphen is methylphenantholine, dmphen is dimethylphenanthroline, tmphen is tetramethylphenanthroline, dmdpphen is dimethyldiphenylphenanthroline, and phen is phenylphenanthroline. In addition, it should be understood that reduced or oxidized forms of these mediators can be used alone or in combination with each other.

Method of the invention

The present invention also provides a method of manufacturing an electrochemical test device of the type described above. This method is schematically illustrated in fig. 6. As shown, a spacer layer 61 is disposed on the first electrode 62. This can be easily achieved using an insulating film or sheet coated with an adhesive on both sides of the spacer layer 61. The spacer layer 61 has an opening 63 through which the first electrode 62 is exposed. This opening may be in the form of a slot 63 as shown in fig. 6, or a groove 73 as shown in fig. 7A and B. An insulating support 64 underlies the electrode 62.

A liquid reagent 65 comprising a redox active species is introduced into opening 63/73 in spacer layer 61 in such a way as to cover at least a portion, but preferably all, of exposed first electrode 62 and at least a portion of side wall 66 of opening 63/73. In one embodiment of the invention, this result is achieved by: opening 63/73 is filled to a sufficient depth to at least partially cover the sidewall. This result can also be achieved by: as the moving droplet on the dispensing nozzle, reagent is applied along the electrode and sidewall such that it leaves a wet trace, or an ink jet or similar dispenser is used in a track to achieve a wetted sidewall and a wetted electrode. It should be noted that in small scale, which is commonly used in glucose testing devices, surface tension pulls the reagent to cover the entire wetted surface, minimizing reagent surface area. This helps the reagent to spread into the corners and up the walls, if wetted already.

The liquid reagent 65 is then dried in the opening 63/73 to form a dry reagent that is disposed in a layer covering the first electrode 62 and at least a portion of the sidewall 66. Drying may be achieved by: the structure is simply allowed to dry in air, dried in an applied air stream, heated, dried in a heated air stream, dried in a vacuum, or dried in a heated vacuum. A sheet material 67 having a conductive electrode surface 68 is then applied on top of the spacer sheet to form a second electrode, which faces the first electrode 62 across the opening 63. The sample inlet opening to the sample receiving space can be created by trimming the intermediate structure so formed transversely through opening 63/73 and into contact with the electrodes for connection to an external meter. Preferred methods of forming the device are described in U.S. provisional patent 60/521,555 and U.S. patent application 10/908,656 (filed 5/20/2005), which are incorporated herein by reference.

Examples

Two devices were constructed using gold-facing electrodes and reagents comprising glucose oxidase, ferricyanide, buffer salts and soluble stabilizers. In the device according to the invention, the reagent extends up the side wall into the vicinity of the second electrode. In the comparative device, the reagent was placed only on the first electrode. As soon as the sample entered the test strip (t ═ 0), 300mV voltage was applied to each device (positive electrode was the first electrode with reagent; negative electrode was the second electrode without reagent). As shown in fig. 2, in the comparative device, there was no real current for an initial short time, and the current started to increase at about 1 second. The current fails to increase significantly until the reagent is able to dissolve and diffuse to the second electrode, thereby providing a reverse reaction that allows current to flow. Fig. 4 shows a current profile of a device according to the invention. In this case, the reagent dissolves and diffuses almost as soon as the sample enters the test strip, and current begins to flow almost immediately (t ═ 0). Thus, at about 3 seconds, a steady state current is achieved in the sandwich geometry electrode structure of this device, and conversely, for another device, about 5 seconds.

Claims (17)

1. An electrochemical test device, comprising:

a bottom substrate having a first electrode disposed thereon, a top substrate having a second electrode disposed thereon, and a spacer disposed between the top and bottom substrates and having an opening therein, thereby defining a sample-receiving space having a bottom surface having the first electrode disposed thereon, a top surface opposing the first surface and having the second electrode disposed thereon, and a sidewall formed by an edge of the opening in the spacer; and

a reagent comprising a redox active species which is oxidised at the first electrode and reduced at the second electrode when the device is in use;

wherein, prior to introducing the liquid sample, in the assay device, the reagent is disposed in a layer covering at least a portion of the first electrode or the second electrode and at least a portion of the sidewall.

2. The device of claim 1, wherein the reagent covers at least 25% of the height of the sidewall.

3. The device of claim 1, wherein the reagent covers at least 50% of the height of the sidewall.

4. The device of claim 1, wherein the reagent covers at least 75% of the height of the sidewall.

5. The device of claim 1, wherein the reagent covers 100% of the height of the sidewall.

6. The device of any one of claims 1 to 5, wherein the redox active species is selected from the group consisting of:

cyhaloferrite

[FeIII(CN)5(ImH)]^2-

[FeIII(CN)5(Im)]^3-

[RuIII(NH3)5(ImH)]³⁺

[RuIII(NH3)5(Im)]²⁺

[FeII(CN)5(ImH)]^3-

[RuII(NH3)5(Im)H]²⁺

[(NC)5FeII(Im)RuIII(NH3)5]^-

[(NC)5FeIII(Im)RuIII(NH3)5]⁰

[(NC)5FeII(Im)RuII(NH3)5]^2-

Ferrocene (Fc)

Ferrocene monosulfonate

Ferrocene disulfonate

FcCO₂H

FcCH2CO₂H

FcCH:CHCO₂H

Fc(CH₂)₃CO₂H

Fc(CH₂)₄CO₂H

FcCH₂CH(NH₂)CO₂H

FcCH₂SCH₂CH(NH₂)CO₂H

FcCH₂CONH₂

Fc(CH₂)₂CONH₂

Fc(CH₂)₃CONH₂

Fc(CH₂)₄CONH₂

FcOH

FcCH₂OH

Fc(CH₂)₂OH

FcCH(Me)OH

FcCH₂O(CH₂)₂OH

1，1′-Fc(CH₂OH)₂

1，2-Fc(CH₂OH)₂

FcNH₂

FcCH₂NH₂

Fc(CH₂)₂NH₂

Fc(CH₂)₃NH₂

1，1′-Me₂FcCH₂NH₂

FcCH₂NMe₂

(R)-FcCH(Me)NMe₂

(S)-FcCH(Me)NMe₂

1，2-Me₃SiFcCH₂NMe₂

FcCH₂NMe₃

FcCH₂NH(CH₂)₂NH₂

1，1′-Me₂FcCH(OH)CH₂NH₂

FcCH(OH)CH₂NH₂

FcCH:CHCH(OH)CH₂NH₂

Fc(CH₂)₂CH(OH)CH₂NH₂

FcCH₂CH(NH₂)CH₂OH

FcCH₂CH(CH₂NH₂)CH₂OH

FcCH₂NH(CH₂)₂OH

1，1′-Me₂FcCHOCONHCH₂

FcCH(OH)(CH₂)₂NH₂

1，1′-Me₂FcCH(OH)CH₂NHAc

FcB(OH)₃

FcC₆H₄OPO₃Na₂

Os(4，7-dmphen)₃

Os(3，4，7，8-tmphen)₃

Os(5，6-dmphen)₃

Os(bpy)₃Cl₂

Os(5-mphen)₃

Os(5-Cl-phen)₃

Os(5-NO₂-phen)₃

Os(5-phphen)₃

Os(2，9-dm4，7-dpphen)₃

Ru(4，7-dmphen)₃

Ru(3，4，7，8-tmphen)₃

Ru(5-mphen)₃

Ru(5，6-dmphen)₃

Ru(phen)₃

[Ru(4，4′-diNH₂-bipy)3]²⁺

Os(bpy)₃

Os(dmbpy)₃

Ru(bpy)₃

Ru(4，4’-diNH₂-bpy)₃

Ru(4，4’-diCO₂Etbpy)₃

Os(bpy)₂dmbpy

Os(bpy)₂(HIm)₂

Os(bpy)₂(2MeHIm)₂

Os(bpy)₂(4MeHIm)₂

Os(dmbpy)₂(HIm)₂

Os(bpy)₂Cl(HIm)

Os(bpy)₂Cl(1-MeIm)

Os(dmbpy)₂Cl(HIm)

Os(dmbpy)₂Cl(1-MeIm)

Ru(bpy)₂(5，5’diNH₂-bpy)

Ru(bpy)₂(5，5’diCO₂Etbpy)

Ru(bpy)₂(4，4’diCO₂Etbpy)

Or its complementary redox form (oxidized or reduced).

7. The device of any one of claims 1 to 6, wherein the reagent further comprises glucose oxidase.

8. A method of manufacturing an electrochemical test device comprising the steps of:

(a) forming an underlying substrate having a first electrode disposed thereon;

(b) forming a spacer layer on the underlying substrate, the spacer layer having an opening formed therein through which the first electrode is exposed and a sidewall within the opening;

(c) a liquid reagent comprising a redox active species is introduced into the opening in the spacer layer,

(d) drying the liquid reagent to form a dry reagent, wherein the liquid reagent is introduced into the opening in such a way that when dried a dry reagent layer is formed which covers at least a part of the first electrode and at least a part of the side wall; and

(e) placing a top substrate having a second electrode disposed thereon on a spacer layer, the spacer layer being aligned such that the second electrode faces the first electrode, thereby forming a sample receiving space having a first surface with the first electrode disposed thereon, a second surface opposite the first surface with the second electrode disposed thereon, and a sidewall formed by an edge of an opening in the spacer.

9. The method of claim 8, wherein the dry reagent covers at least 25% of the height of the sidewall.

10. The method of claim 8, wherein the dry reagent covers at least 50% of the height of the sidewall.

11. The method of claim 8, wherein the dry reagent covers at least 75% of the height of the sidewall.

12. The method of claim 8, wherein the reagent covers 100% of the height of the sidewall.

13. A method according to any one of claims 8 to 12, wherein the spacer layer applied in step (b) comprises an adhesive coating and a release sheet, which is placed on the side of the spacer layer facing away from the first electrode, whereby a portion of the side wall consists of the release sheet, the method further comprising the step of removing the release sheet after drying the liquid agent to expose the adhesive layer.

14. The method of claim 13, wherein the liquid agent is introduced into the opening in a volume sufficient to fill the opening to a level such that the sidewall portion formed by the release sheet is at least partially covered.

15. The method of any one of claims 8 to 12, wherein the liquid reagent is introduced in a volume sufficient to fill the opening to a level such that a sidewall portion of the opening is partially covered.

16. The method of any one of claims 8 to 15, wherein the opening is in the form of a slot, the slot being defined by sidewalls on all sides.

17. The method of any one of claims 8 to 15, wherein the opening is in the form of a trench, the trench being bounded only by sidewalls on two opposing sides.