APPARATUS FOR SUPPORTING AN ARRAY OF LAYERS OF AMPHIPHILIC
MOLECULES AND METHOD OF FORMING AN ARRAY OF LAYERS OF AMPHIPHILIC
MOLECULES
The present invention relates to an apparatus for supporting an array of amphiphilic
molecules and a method of forming such an array. In particular, the invention relates to the
efficient formation of arrays of amphiphilic molecules. One area of application is the
preparation of lipid bilayers.
In one type of known technique, a membrane based layer of amphiphilic molecules may
be used as a means of separating two volumes of aqueous solution. The amphiphilic layer resists
the flow of current between the volumes when a potential difference is applied between the two
volumes. A membrane penetrating protein is inserted into the amphiphilic layer to allow the
passage of ions across the layer, which is recorded as an electrical signal detected by electrodes
placed in each of the aqueous solutions, such as disclosed in WO2009/077734.
In this technique, a target analyte may interact with the membrane penetrating protein to
modulate the flow of ions and may be detected by observing the resultant variations in the
electrical signal. This technique therefore allows the layer of amphiphilic molecules to be used
as a biosensor to detect the analyte.
The layer of amphiphilic molecules has a two-fold purpose in this technique. Firstly, the
layer provides a platform for the protein that acts as a sensing element. Secondly, the layer
isolates the flow of ions between the volumes. The electrical resistance of the layer ensures that
the dominant contribution of ionic flow in the system is through the membrane protein of
interest, with negligible flow through the layer of amphiphilic molecules, thus allowing detection
with single protein channels.
A specific application of this technique is in nanopore sensing, where the number of
membrane proteins is kept small, typically between 1 and 100, so that the behaviour of a single
protein molecule can be monitored electrically. This method gives information on each specific
molecular interaction and hence provides richer information than a bulk measurement.
However, due to the small currents involved, typically a few pA, this approach relies on the
formation of a very high resistance seal, typically greater than 1GQ, and sufficient electrical
sensitivity to measure the current.
While the requirements for stochastic sensing have been met in the laboratory, conditions
and expertise limit its practical application in commercial products. In addition, laboratory
methods are laborious and time-consuming and are not scalable easily to the high-density arrays
that are desirable for any commercial biosensor. Furthermore, the fragility of single amphiphilic
layer membranes means that they can be difficult to form, so that anti-vibration tables are often
employed in the laboratory. Necessitating the use of such anti-vibration tables would not be
desirable in a commercial product.
There have been great efforts to increase the ease of bilayer formation using micro
fabrication. Some techniques have attempted to miniaturise standard systems for folded lipid
bilayers or painted lipid bilayers. Other techniques include bilayer formation on solid substrates
or directly on electrode surfaces, through either absorption or adsorption. A large proportion of
nanopore sensing devices form a bilayer by using a variant of either the folded lipid bilayers
technique, or the painted bilayer technique. To date, most have concentrated either on novel
methods of aperture formation, on utilising the emerging technologies in micro fabrication to
miniaturise the device, or to create a plurality of addressable sensors such as disclosed in
EP2107040 and WO2010/122293.
There are problems associated with the conventional supported amphiphilic layer
approach that makes the approach unsuitable. The first problem lies with the resistance of the
lamellar membrane which typically is about 100MW. While this may be suitable for examining
protein behaviour at large protein concentrations, it is not sufficient for a high-fidelity assay
based on single molecule sensing. To achieve single-molecule sensing a resistance of at least
1GQ, and for some applications one or two orders of magnitude higher, is required. The second
problem relates to the small volume of solution trapped in the small distance between the
amphiphilic layer and the solid support, typically of the order of lnm. This small volume does
not contain many ions, and this affects the stability of the potential across the amphiphilic layer
and limits the duration for which recording can be performed.
The techniques used in the silicon chip industry provide an attractive technology for
creating a large number of electrodes that could be used in biosensor applications. This
approach is disclosed in the related applications US 7,144,486 and US 7,169,272. US 7,144,486
discloses a method of fabricating a microelectrode device containing microcavities etched into
layers of an insulator material. The devices are said to have a wide range of electrochemical
applications in which electrodes in the cavities allow measurement of electrical signals.
In summary, the known technologies discussed above either present methods of
amphiphilic layer formation that cannot reproducibly achieve high resistances; suffer from low
ionic reservoirs; are not capable of high duration direct current measurements; and/or require a
separate fluidic chamber for each array element. This limits the scale up of the techniques to
produce a high-density array device.
WO 2009/077734 describes a simplified apparatus to prepare amphiphilic layers across a
recess and to scale the apparatus with multiple recesses forming chambers of a large scale sensor
array without any need for a complicated apparatus.
In this method a lipid amphiphilic layer is formed as a layer separating two volumes of
aqueous solution, the method comprising: (a) providing an apparatus comprising elements
defining a chamber, the elements including a body of non-conductive material having formed
therein at least one recess opening into the chamber, the recess containing an electrode; (b)
applying a pre-treatment coating of a hydrophobic fluid to the body across the recess; (c) flowing
aqueous solution, having amphiphilic molecules added thereto, across the body to cover the
recess so that aqueous solution is introduced into the recess from the chamber and so that a layer
of the amphiphilic molecules forms across the recess separating a volume of aqueous solution
A key feature of this method is the preparation of high quality amphiphilic layers that are
suitable for high sensitivity biosensor applications such as nanopore sensing and single channel
recording. The method has been demonstrated to form amphiphilic layers of high resistance,
providing highly resistive electrical seals having an electrical resistance of greater than 1GQ,
typically lOOGQ, which for example, enable high-fidelity recordings from single protein pores.
In this method, formation of a layer of the amphiphilic molecules across a recess simply
by flowing the aqueous solution across the body to cover the recess is possible provided that a
pre-treatment coating of a hydrophobic fluid is applied to the body across the recess. The pretreatment
coating assists formation of the amphiphilic layer and aids the wetting of the,
microcavity forming the sensor well, with aqueous solution.
However, under some circumstances the formation of high quality amphiphilic layers
may be compromised. The present invention aims to at least partly address this problem.
According to a first aspect of the invention there is provided an apparatus for supporting
an array of layers of amphiphilic molecules, the apparatus comprising: a body, formed in a
surface of the body, an array of sensor wells capable of supporting a layer of amphiphilic
molecules across the sensor wells, the sensor wells each containing an electrode for connection
to an electrical circuit, and formed in the surface of the body between the sensor wells, flow
control wells capable of smoothing the flow of a fluid across the surface.
This aspect is directed to a body in which inactive flow control wells are provided for
increasing uniformity of distribution. That is, the additional wells reduce any stick/slip
characteristics, resulting in a more predictably uniform wetted surface. The provision of the
additional wells allows the sensor wells to be distributed and function as desired, without
needing to account for the wetting characteristics of the system. That is, the desired sensor well
distribution can be selected, and the additional wells can be supplied to account for the system
wetting characteristics.
Optionally, the cross-sectional area of a flow control well is less than the area of a sensor
well.
Optionally, the flow control wells do not contain electrodes. Alternatively, the flow
control wells each contain an electrode, the electrodes in the sensor wells being connected to the
electrical circuit but the electrodes in the flow control wells not being connected to the electrical
circuit. In the embodiments where the flow control wells do not function as active sensor wells,
their function is to solely improve the wetting characteristics of the system. As such the
constraint of requiring the flow control wells to be able to function as flow sensor wells is
removed and the flow control wells may be provided of dimensions, for example the crosssectional
area of the aperture or shape of the wells, or of a pitch that are/is unsuitable for use as
sensor wells.
Optionally, the apparatus further comprises a cover over the surface of the body defining
a cavity therebetween, and a common electrode arranged in the cavity for connection to the
electrical circuit. The cover can have an internal surface facing the surface of the body that is
roughened to smooth the flow of fluid thereover.
Optionally, the array of sensor wells is a regular array, and the flow control wells consist
of a regular array of flow control wells. Optionally, the pitch of the array of at least a portion of
the flow control wells is smaller than the pitch of at least a portion of the array of sensor wells.
That is, the axial distance between the flow control wells can be smaller than the axial distance
between the sensor wells. The flow control wells may be of a different dimension than the flow
sensor wells, for example a different size, a different cross-sectional area and/or a different
cross-sectional area of the aperture than the sensor wells. Optionally, the sensor wells are
circular, and optionally the flow control wells are square. Optionally, the flow control wells are
distributed over a larger area than the sensor wells.
Optionally, the sensor wells and flow control wells are arranged such that a pretreatment,
being a fluid capable of interacting with the amphiphilic molecules, on the surface
would not enter the Cassie-Baxter state. Optionally, the sensor wells and flow control wells are
shaped to provide a surface roughness r, defined as the total area of the surface and wells divided
by the projected area of the surface, and a solid surface area fraction / , defined as the area of the
surface between the wells divided by the projected area of the surface, that meet the requirement
in respect of a pre-treatment, that is a fluid capable of interacting with the amphiphilic
molecules, having a contact angle Qthat {{f -\)l{r- ^))>cos(9). This ensures that the pretreatment
can enter the wells.
Optionally, the wells are formed on the surface with a number density of 3.2xl0 5
wells/micron 2 or more, optionally 6.4xl0 5 wells/micron 2 or more, further optionally 1.5xl0 4
wells/micron 2 or more, and still further optionally 2.5xl0 4 wells/micron 2 or more.
Optionally, the apparatus further comprises a pre-treatment of a hydrophobic fluid
applied to the surface of the body.
According to this aspect, there is also provided a method of preparing an apparatus for
forming an array of amphiphilic layers , the method comprising: providing an apparatus as
discussed above; delivering across the surface of the body to the wells a pre-treatment coating of
a hydrophobic fluid. The pre-treatment coating serves to support the amphiphilic layer such that
a highly resistive electrical seal may be formed across the well.
Optionally, the pre-treatment is delivered in a solvent, the method further comprising
drying the surface of the body to remove the solvent. Said step of drying the surface of the body
to remove the solvent is preferably performed under a pressure below atmospheric pressure.
Optionally, the method is performed so that each of the following conditions is met: the
visible coverage of the surface by the pre-treatment is less than 15% of the area in which the
array of sensor wells is located; the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values. Further optionally, the method
is performed so that each of the following conditions is met: the visible coverage of the surface
by the pre-treatment is less than 5% of the area in which the array of sensor wells is located; the
proportion of sensor wells that are filled is less than 0.5%; and the values of rectangularity and
the perimeter of all the annuli of pre-treatment around the respective sensor wells falls within a
2 0% of the average values. In this context, the 'average values' refer to the mean values of the
rectangularity and the perimeter of the annuli, respectively, as calculated for all the sensor wells.
According to this aspect, there is also provided a method of forming an array of the
sensor wells each containing an electrode for connection to an electrical circuit, wherein the
wells have a number density of 3.2xl0 5 wells/micron 2 or more, optionally 6.4xl0 5
wells/micron 2 or more, further optionally 1.5xl0 4 wells/micron 2 or more, and still further
optionally 2.5xl0 4 wells/micron 2 or more.
Optionally, all the wells are sensor wells. Alternatively, some of the wells are sensor
wells, and the remainder of the wells are flow control wells, formed in the surface of the body
between the sensor wells.
Optionally, the area of a flow control well is less than the area of a sensor well.
Optionally, the flow control wells do not contain electrodes. Alternatively, the flow
control wells each contain an electrode, the electrodes in the sensor wells being connected to the
electrical circuit but the electrodes in the flow control wells not being connected to the electrical
circuit.
Optionally, the array of sensor wells is a regular array, and the flow control wells consist
of a regular array of flow control wells. Optionally, a pitch of the array of flow control wells is
smaller than a pitch of the array of sensor wells. Optionally, the sensor wells are circular, and
the flow control wells are square.
Optionally, the flow control wells are distributed over a larger area than the sensor wells.
Optionally, the apparatus further comprises a cover over the surface of the body defining
a cavity therebetween, and a common electrode arranged in the cavity for connection to the
electrical circuit. Optionally, the cover has an internal surface facing the surface of the body that
is roughened to smooth the flow of fluid thereover.
Optionally, the wells have an area density of 0.141 or more.
Optionally, the wells are arranged such that a pre-treatment applied to the surface of the
body does not enter the Cassie-Baxter state. Optionally, the sensor wells and flow control wells
are shaped to provide a surface roughness r, defined as the total area of the surface and wells
divided by the projected area of the surface, and a solid surface area fraction / , defined as the
area of the surface between the wells divided by the projected area of the surface, that meet the
requirement in respect of a pre-treatment, that is a fluid capable of interacting with the
amphiphilic molecules, having a contact angle Qthat {{f -\)l{r- ^))>cos(9).
Optionally, the apparatus further comprises a pre-treatment of a hydrophobic fluid that is
applied to the sensor wells.
According to the second aspect, there is also provided a method of preparing an apparatus
for forming an array of sensor wells, the method comprising: providing an apparatus of the
second aspect, as discussed above; delivering across the surface of the body to the wells a pretreatment
of a hydrophobic fluid.
Optionally, the pre-treatment is delivered in a solvent, the method further comprising
drying the surface of the body to remove the solvent. Optionally, the step of drying the surface
of the body to remove the solvent is performed under a pressure below atmospheric pressure.
Optionally, the method is performed so that each of the following conditions is met: the
visible coverage of the surface by the pre-treatment is less than 15% of the area in which the
array of sensor wells is located; the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values. Further optionally, the method
is performed so that each of the following conditions is met: the visible coverage of the surface
by the pre-treatment is less than 5% of the area in which the array of sensor wells is located; the
proportion of sensor wells that are filled is less than 0.5%; and the values of rectangularity and
the perimeter of all the annuli of pre-treatment around the respective sensor wells falls within a
2 0% of the average values.
According to the second aspect, there is also provided a method of forming an array of
layers of amphiphilic molecules, the method comprising: preparing an apparatus by a method of
the second aspect, as discussed above; and flowing a fluid containing amphiphilic molecules
across the surface of the body to form layers of amphiphilic molecules across at least some of the
array of sensor wells.
According to a third aspect, there is provided a method of preparing an apparatus for
forming an array of layers of amphiphilic molecules, the method comprising: providing an
apparatus comprising a body, and, formed in a surface of the body, an array of wells, at least
some of which are sensor wells capable of supporting a layer of amphiphilic molecules across
the sensor wells after application to the sensor wells of a pre-treatment of a hydrophobic fluid,
the sensor wells each containing an electrode for connection to an electrical circuit, and
delivering across the surface of the body a pre-treatment, that is a fluid capable of interacting
with the amphiphilic molecules, in a solvent to apply the pre-treatment to the wells; and drying
the surface of the body to remove the solvent under a pressure below atmospheric pressure.
According to this aspect, the use of low-pressure drying produces a more uniform dried
pre-treatment on the surface of the body.
Optionally, the apparatus is an apparatus according to the first or second aspect,
discussed above.
Optionally, the method can be performed so that each of the following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 15%> of the area in which the
array of sensor wells is located; the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values. Further optionally, the method
can be performed so that each of the following conditions is met: the visible coverage of the
surface by the pre-treatment is less than 5% of the area in which the array of sensor wells is
located; the proportion of sensor wells that are filled is less than 0.5%; and the values of
rectangularity and the perimeter of all the annuli of pre-treatment around the respective sensor
wells falls within a 20% of the average values.
The third aspect of the invention also provides a method of forming an array of layers of
amphiphilic molecules, the method comprising: preparing an apparatus by a method according to
the method of the third aspect, as discussed above; and flowing a fluid containing amphiphilic
molecules across the surface of the body to form layers of amphiphilic molecules across at least
some of the array of sensor wells.
According to a fourth aspect of the invention, there is provided a method of preparing an
apparatus for forming an array of layers of amphiphilic molecules, the method comprising:
providing an apparatus comprising a body, and, formed in a surface of the body, an array of
wells, at least some of which are sensor wells capable of supporting a layer of amphiphilic
molecules across the sensor wells after application to the sensor wells of a pre-treatment of a
hydrophobic fluid, the sensor wells each containing an electrode for connection to an electrical
circuit, and delivering to the body a pre-treatment of a hydrophobic fluid, the method being
performed so that each of the following conditions is met: the visible coverage of the surface by
the pre-treatment is less than 15% of the area in which the array of sensor wells is located; the
proportion of sensor wells that are filled is less than 5%; and the values of rectangularity and of
the perimeter of each of the annuli of pre-treatment around the respective sensor wells falls
within 40% of the average values. The visible coverage can be determined with any appropriate
light-source. For example, under appropriate lighting conditions, the coverage may be visible in
normal light. Alternatively, additives in the pre-treatment may be used to highlight the coverage
under particular lighting conditions. For example, in one embodiment of the invention, a green
fluorescent dye (a boron-dipyrromethene) is used to highlight the pre-treatment and a red
fluorescent dye (sulforhodamine) is used to highlight the buffer under the membrane layer.
Optionally, the method is performed so that each of the following conditions is met: the
visible coverage of the surface by the pre-treatment is less than 5% of the area in which the array
of sensor wells is located; the proportion of sensor wells that are filled is less than 0.5%; and the
values of rectangularity and the perimeter of all the annuli of pre-treatment around the respective
sensor wells falls within a 20% of the average values.
Optionally, the pre-treatment is applied to the body in a solvent, and the method further
comprises drying the surface of the body to remove the solvent, the method being performed so
that said conditions are met after said drying.
The fourth aspect further provides a method of forming an array of layers of amphiphilic
molecules, the method comprising: preparing an apparatus according to the method of the fourth
aspect, discussed above; and flowing a fluid containing amphiphilic molecules across the surface
of the body to form layers of amphiphilic molecules across at least some of the array of sensor
wells.
The fourth aspect further provides an apparatus for forming an array of layers of
amphiphilic molecules, the apparatus comprising: a body; and formed in a surface of the body,
an array of wells, at least some of which are sensor wells capable of supporting a layer of
amphiphilic molecules across the sensor wells after application to the sensor wells of a pretreatment
of a hydrophobic fluid, the sensor wells each containing an electrode for connection to
an electrical circuit, the array of wells being arranged such that after delivery to the body of a
pre-treatment that is a fluid capable of interacting with the amphiphilic molecules, each of the
following conditions is met: the visible coverage of the surface by the pre-treatment is less than
15% of the area in which the array of sensor wells is located; the proportion of sensor wells that
are filled is less than 5%; and the values of rectangularity and the perimeter of all the annuli of
pre-treatment around the respective sensor wells falls within a 40% of the average values.
Optionally, the array of wells is arranged such that after delivery to the body of a pretreatment
of a hydrophobic fluid, each of the following conditions is met: the visible coverage of
the surface by the pre-treatment is less than 5% of the area in which the array of sensor wells is
located; the proportion of sensor wells that are filled is less than 0.5%; and the values of
rectangularity and the perimeter of all the annuli of pre-treatment around the respective sensor
wells falls within a 20% of the average values.
Optionally, the apparatus further comprises a pre-treatment of a hydrophobic fluid
applied to the sensor wells.
The present invention will be described with reference to exemplary embodiments and
the accompanying Figures in which:
Fig.l is a diagram of ideal fluid behaviour in a well;
Fig. 2 is a diagram of undesirable fluid behaviour in a well;
Fig. 3 is a diagram showing different wetting behaviours;
Figs. 4a and 4b are of the expected modified contact angles for different 'native' contact
angles, for an array of 50 micron wells spaced (a) 63 microns apart and (b) 8 1 microns apart;
Figs. 5a - c are images that illustrate how changing surface design affects pre-treatment
dispersal;
Fig 6 is an image showing pre-treatment dispersal for a first design;
Figs. 7a-d are images showing pre-treatment dispersal for a second design;
Figs. 8a are images showing pre-treatment dispersal for the second design under different
conditions;
Figs. 9a-9c are images showing pre-treatment dispersal for a third design;
Fig. 10a is a schematic representation of a fourth design, and Figs. lOb-c are images
showing pre-treatment dispersal for the fourth design;
Fig. 1l a is a schematic representation of a fifth design, and Figs. 1lb-c are images
showing pre-treatment dispersal for the fifth design;
Fig. 12a is a schematic representation of a sixth design, and Figs. 12 b and 12c are
images showing pre-treatment dispersal for the sixth design; and
Fig. 13a is a schematic representation of a sixth design, and Figs. 13 b and 13c are
images showing pre-treatment dispersal for the seventh design;
As mentioned above, the techniques of WO 2009/077734, herein incorporated by
reference in its entirety, can result in amphiphilic layers of compromised quality in some
circumstances. The present invention has identified that this can be the result of the pretreatment
coating being, in some parts of the array, either greater or less than an optimal level.
Fig. 1 shows a schematic cross section through a microcavity or sensor well 10 of a
sensor array. The well 10 is formed in a material 11 such as SU-8 forming a body, and many
wells 10 may be formed in close proximity within the material to form an array of sensor wells.
Preferably the material in which the wells are formed is itself solid and not porous, so that the
wells maintain their integrity and liquid does not leak or leach from the wells. The body may
also be made of other materials such as such as a positive or negative photoresists, plastics such
as polycarbonate or polyester or solid state inorganic materials such as silicon, glass or silicon
nitride. Examples of photoresists that may be used are SU8 2000 or 3000 series materials,
poly(methyl methacrylate) (PMMA), poly(methyl glutarimide) (PMGI), phenol formaldehyde
resin (DNQ/Novolac), or polyhydroxystyrene-based polymers. At the bottom of the well is an
electrode 12 for connection to an electrical circuit, which can be used (in combination with
another electrode above the well, not shown in Fig. 1) to monitor the flow of current through the
well 10.
In practice, an array of such sensor wells 10 formed in a body will be provided in an
apparatus further comprising a cover over the surface of the body, so as to define a cavity
between the cover and the body. An electrode is arranged in the cavity for connection to the
electrical circuit, and acts a common electrode for the wells in the array.
Fig. 1 shows the ideal configuration, in which a pre-treatment 20 is pinned tightly to the
edges of the well 10. This configuration allows for the maximum trapped volume (i.e. volume of
amphiphilic layer 30) within the well 10. This configuration results in a biosensor with the
longest lifetime.
The pre-treatment is a fluid capable of interacting with the amphiphilic molecules. The
pre-treatment coating is typically a hydrophobic substance, usually having long chain molecules,
in an organic solvent. Suitable organic substances include without limitation: n-decane,
hexadecane, isoecoisane, squalene, pristane (2,6,10,14-tetramethylpentadecane), fluorinated oils
(suitable for use with fluorinated lipids), alkyl-silane (suitable for use with a glass membrane)
and alkyl-thiols (suitable for use with a metallic membrane). Suitable solvents include but are
not limited to: pentane, hexane, heptane, octane, decane, and toluene. The material might
typically be 0 .1mΐ to 1Omΐ of 0.1% to 50% (v/v) hexadecane in pentane or another solvent, for
example 2m1of 1% (v/v) hexadecane in pentane or another solvent, in which case lipid, such as
diphantytanoyl-sn-glycero-3-phosphocholine (DPhPC), might be included at a concentration of
0.6mg/ml.
Some specific materials for the pre-treatment coating 30 are set out in Table 1 by way of
example and without limitation.
Table 1 : Examples of pre-treatment materials.
The amphiphilic layer can be made of any amphiphile that forms a lamellar phase.
Amphiphiles include lipids capable of forming lipid bilayers. The amphiphiles are chosen such
that an amphiphilic layer having the required properties, such as surface charge, ability to
support membrane proteins, packing density or mechanical properties, is formed. The
amphiphiles can comprise one or more different components. For instance, the amphiphiles can
contain up to 100 amphiphiles. The amphiphiles may be naturally-occurring or synthetic . The
amphiphile may be a block copolymer.
In embodiments where the amphiphile is a lipid, the lipid typically comprises a head
group, an interfacial moiety and two hydrophobic tail groups which may be the same or
different. Suitable head groups include, but are not limited to, neutral head groups, such as
diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, such as
phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM); negatively
charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS),
phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); and positively charged
headgroups, such as trimethylammonium-Propane (TAP). Suitable interfacial moieties include,
but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or
ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to,
saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic acid), myristic acid (n-
Tetradecononic acid), palmitic acid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and
arachidic (n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (cis-9-
Octadecanoic); and branched hydrocarbon chains, such as phytanoyl. The length of the chain
and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary.
The length of the chains and the position and number of the branches, such as methyl groups, in
the branched hydrocarbon chains can vary. The hydrophobic tail groups can be linked to the
interfacial moiety as an ether or an ester.
The lipid can also be chemically-modified. The head group or the tail group of the lipid
may be chemically-modified. Suitable lipids whose head groups have been chemically-modified
include, but are not limited to, PEG-modified lipids, such as l,2-Diacyl-sn-Glycero-3-
Phosphoethanolamine-N -[Methoxy(Polyethylene glycol)-2000]; functionalised PEG Lipids,
such as l,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(Poly ethylene
Glycol)2000]; and lipids modified for conjugation, such as l,2-Dioleoyl-sn-Glycero-3-
Phosphoethanolamine-N-(succinyl) and l,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-
(Biotinyl). Suitable lipids whose tail groups have been chemically-modified include, but are not
limited to, polymerisable lipids, such as l,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-
Phosphocholine; fluorinated lipids, such as l-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-
Phosphocholine; deuterated lipids, such as l,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine;
and ether linked lipids, such as l,2-Di-0-phytanyl-sn-Glycero-3-Phosphocholine.
The lipid may comprise one or more additives that will affect the properties of the lipid
bilayer. Suitable additives include, but are not limited to, fatty acids, such as palmitic acid,
myristic acid and oleic acid; fatty alcohols, such as palmitic alcohol, myristic alcohol and oleic
alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol and stigmasterol;
lysophospholipids, such as l-Acyl-2-Hydroxy-sn- Glycero-3 -Phosphocholine; and ceramides.
The lipid preferably comprises cholesterol and/or ergosterol when membrane proteins are to be
inserted into the amphiphilic layer.
When pre-treatment oil 20 is not deposited in the optimum configuration of Fig. 1, a
smaller trapped volume is the most probable outcome. There is also a higher probability that
excess pre-treatment oil will be located on the upper surface of the well 10. This is shown
schematically in Fig. 2 . In Fig. 2, the pre-treatment 20 is not pinned tightly to the edges of the
well 10. As a result, the trapped volume of the amphiphilic molecule 30 is reduced. Further,
pre-treatment 20a is also present on the upper surface of the well 10.
In order to form a good contact between pre-treatment 20 and the amphiphilic layer 30, it
is preferable to use a hydrophobic material for forming the well 10. This encourages a small
contact angle between the pre-treatment 20 and the amphiphilic layer 30. However, this also
makes it more likely that pre-treatment oil will form droplets 20a on the surface of the array
material unless pinned into the well 10 and collected by Laplace pressures. The appropriate
hydrophobic surface properties may be achieved by suitable selection of materials. However,
where there are conflicting constraints, for example where the desired surface properties are not
available using photoresist material appropriate for fabrication of the required structure, this may
not be possible. In this case, commonly, surface treatments are applied to achieve a hydrophilic
surface, such as the addition of a chemical coating or plasma modification. These methods are
not ideal, typically they are unstable over a long product storage lifetime or may cause
interference with the sensor chemical system.
Where there is a desire to form the amphiphilic layers quickly, requiring fast flow rates
over the surface or where a very large scale array is used, it has been found that the flow of
aqueous solution during the amphiphilic layer formation phase may cause a transfer of pretreatment
20 to the downstream areas of the array or lead to the creation of an emulsion in the
aqueous solution, which is undesirable.. This is more likely in situations where pre-treatment oil
20 is located outside of the well, for example on the SU-8 surface.
In the current invention, the introduction of surface patterning to the bulk surface of the
array allows for improved formation of the pre-treatment layer 20 with good uniformity and aids
retention of the pre-treatment layer during the subsequent fluid flow associated with amphiphilic
layer formation.
The uniformity of pre-treatment distribution can be further enhanced by extending the
surface patterning beyond the bulk surface of the array to consider the other internal faces of the
fluidic flow cell in which the array is contained. In this example, during the pre-treatment
application phase, the pre-treatment oil material is also coated onto all other internal surfaces.
During the subsequent fluid flow steps this material may also be redistributed, therefore
compromising formation of high quality lipid amphiphilic layers. A surface pattern can be
introduced to these other surfaces, and tailored to control the degree of coating with pretreatment
and to enhance retention of the pre-treatment on those surfaces enhancing the overall
performance of the apparatus.
The surface patterning also enables the required surface hydrophobicity, which is
conventionally achieved by surface chemistry modification of the array material, to be achieved
through altering the ratio of contribution of surface energies between that of the native material
and that of air, or whatever the surrounding bulk medium may be.
The surface states that may exist for a well-containing surface are defined by the overall
thermodynamic position.
In the 'Cassie-Baxter' state, the hydrophobicity is high enough that the wells are not
filled by the wetting fluid, but remain filled with the bulk medium. However, this state is
thermodynamically unstable and can, under the correct circumstances, collapse to a lower energy
state.
In the most thermodynamically stable 'Wenzel' state, the wells are completely filled by
the wetting fluid. Once achieved it is impossible to revert between the Wenzel and Cassie-
Baxter states.
Fig. 3 illustrates the wetting of (a) a flat surface in comparison to wetting a surface
containing microstructures in the (b) Wenzel and (c) Cassie-Baxter states. As can be seen from
the Figure, the contact angle Qdiffers in the different states.
The modified angles of the Wenzel, q , and Cassie-Baxter, 6 c , states can be calculated
once the contact angle, 6 , of the native material is known.
= p c s - ) —
= c s
where f is defined as the area of the surface between the wells divided by the projected
area of the surface (calculated as: (total area - well area)/(total area)), r is defined as the ratio of
true area of the solid surface to the apparent area.
As such it is possible to calculate the effects for both phenomena over a range of fluid
contact angles.
Figs. 4a and 4b show graphs of the expected modified contact angles for different
'native' contact angles, for an array of 50 micron wells spaced (a) 63 microns apart and (b)
8 1 microns apart. These graphs show that there is a significant difference in Cassie-Baxter
contact angles between surfaces of 50 m micro-wells spaced either 63 or 8 1 m apart. For
example, native SU-8 has a contact angle of around 76°, thus, for 63 m wells, a Wenzel state
would have a modified contact angle of around 65°, whilst the Cassie-Baxter state exhibits
modified contact angles in the region of 115°.
As such the surface properties of the array can thus be tailored to specific fluids or to
produce a desired surface state, by controlling the surface patterning. In particular, it may be
desirable to form the array with additional wells, not intended for sensing, in order to modify the
surface properties. Such additional wells may be inactive for sensing, either because they do not
contain an electrode or because the electrode is not connected to the sensing circuitry. This
approach holds several advantages.
For flow through pre-treatment application on the large-scale, the surface can be
controlled to promote pinning of the pre-treatment on the sensor array surface so that pretreatment
does not move during amphiphilic layer formation. Additionally, it is preferable to
avoid the Cassie-Baxter state, otherwise the pre-treatment will not fill the wells. That is, it is
preferable to design the surface to have a contact angle Qfor which: {{f -l)/(r- ^))>cos(9).
Using a high density of wells, including inactive wells, over the bulk surface forming the
surface pattern also allows maximum flexibility into the design. That is, if it becomes desirable
to change the arrangement of the sensing wells, for example to produce a more closely packed
electronic array, this can be produced with minimal impact to the overall surface by appropriate
'balancing' with inactive wells. That is, the inactive wells can be formed in the surface of the
body in which the 'active' wells have been formed, adding to the array of active wells to create
the desired surface properties. As a result, the surface properties can remain virtually unaltered
whilst varying the structure of the active array, and so the optimal fluidic procedure will not need
to be changed. The additional 'flow control' wells may not contain electrodes, or may contain
electrodes that are not attached to the electrical circuit of the sensor wells.
Controlling the hydrophobicity based on the well geometry and placement avoids the
need for additional processing steps associated with modifying the surface properties by
chemical means. Further, this method of surface control is applicable to all materials, making it
unnecessary to tailor a particular chemistry to a particular material.
In addition, it has been found that the flow through of pre-treatment is also enhanced by
using micro-patterned surfaces. The pre-treatment front can be observed to progress across an
array more smoothly in the presence of additional wells, particularly on larger arrays. That is,
the additional wells increase the homogeneity of flow across the surface of the body such that the
uniformity of wetting is increased. The additional wells are capable of increasing the uniformity
of the distribution of said pre-treatment during deliver across the surface of the body. This
smoothing reduces the tendency for the fluid to undergo large scale pinning during flow which
results in so-called 'stick/slip' movement of a fluid front. Wetting in this stick/slip fashion is
irregular and can result in the fluid being pinned for a period of time before moving to the next
pinning position. This can also result in de-wetting of surfaces that have already been wetted as
the shape of the wetting profile changes. To this end, it can also be preferable to roughen the
internal surface of the cover, opposite the body, to further smooth the flow of fluid. It can also
be preferable to provide the additional wells over a large area than the sensor wells, in order to
ensure the edges of the array of sensor wells experience the enhanced flow of pre-treatment.
The pre-treatment distribution is monitored by tagging the pre-treatment oil with a
fluorescent dye. The dye is then imaged using epi-fluorescence microscopy in situ.
The images show in Figs. 5a-d show an example of the difference in distributions
obtained by introducing additional wells in a surface for otherwise identical fluidic flows of pretreatment
oils dissolved in hexane over an array. Fig. 5a shows an overview of a treated array of
active wells, with no additional wells, whilst Fig. 5b shows a close up view of some the wells.
The bright areas indicate the presence of pre-treatment. As is particularly clear in Fig. 5b, many
of the wells are completely filled by pre-treatment, and there is much excess pre-treatment on the
surface. In contrast, Fig. 5c shows an overview of an array incorporating additional (smaller)
inactive wells in addition to the active wells, and Fig. 5d is a close up view of some of the wells.
The pre-treatment uniformly forms the 'ideal' ring structure around the wells and no wells are
completely filled. Further, there is much less variation between wells in the quality of pretreatment
(even only considering non-filled wells). It is noted that the brighter areas towards the
right hand side of Fig. 5c is excess pre-treatment located on the window of the cell not on the
array surface (focal plane).
These images illustrate that the behaviour of fluid flowing over a surface containing wells
can be influenced by changing the surface texture in between the wells. The introduced wells
may, but need not, also be used as active wells. As such, if it is desired to keep a certain active
well spacing, but improve the distribution of the pre-treatment, that is possible by introducing
'inactive' wells. These inactive wells help the pre-treatment flow across the surface during the
application stage and further aid in the formation of a well distributed pre-treatment during the
drying phase.
Exemplary experiments are discussed below.
Experimental procedures
Materials Required:
Clean room, Oven, RTE, Hotplate x2, Mask aligner, Resist spinner, Develop dishes x2,
Nitrogen supply, Wafer tweezers, Inspection microscope, Silicon Wafers, SU-8 10 photoresist,
SU-8 2 photoresist, EC Developer, Photolithography mask 1st layer: 4KCSH51 4201,
Photolithography mask 2nd layer: 4KCSH41 4149, Acetone (propan-2-one), IPA (propan-2-ol /
2-propanol).
Method for preparing wafers with well designs:
To ensure that the surfaces were clean from organic greases and salts from manufacturing
and handling, silicon wafers were rinsed with acetone, 2-propanol and deionised water prior to
use. The wafers were dried with a gentle supply of nitrogen. Wafers were then placed in a
preheated oven for 1 hour at 150°C. The SU-8 solutions (SU-8 2, and SU-8 10) were removed
from cold storage and allowed to reach room temperature prior to use. The hotplates were
cleaned and allowed to reach stable temperatures of 80°C and 110°C. The spin coater and
developer dishes were set-up ready for use. SU-8 2 (9mL)was spun onto oxygen plasma treated
(200W, 50mTorr) wafers at 2000 rpm, which was then first placed on a hotplate at 80°C for 1
minute prior to a 2 minute treatment on a hotplate set to 110°C. The soft-baked SU-8 2 layer was
then exposed to the electrode-mask for 10 seconds, after suitable alignment to the wafer. A post
exposure bake at 80°C for 1 minute and 2 minutes at 110°C for 2 minutes was performed. The
wafer was then developed in a two-stage rinsing process, followed by a thorough rinse with 2-
propanol. The wafer was dried with nitrogen prior to inspection. The wafer was then re-spun
with SU-8 10 (9mL) at 1600 rpm. The wafer was then baked again at 80°C for 1 minute
followed by 2 minutes at 110°C. The wafer was then aligned and exposed to UV for 55 seconds
under the mask. A further post exposure bake of 3 minute at 80°C followed by a second at
110°C for 7 minutes was performed. The wafer was then developed thoroughly and washed with
2-propanol prior to a de-scumming oxygen plasma process of 1 minute. The wafers were then
hard-baked at 150°C for 1 hour. Wafers were then processed for dicing and bonding.
Diced and bonded 128 chips were then examined for surface defects prior to use. A
single water wash removed surface dust particles, whilst a single ethanol wash removed surface
greases prior to use.
Designs were fabricated on S1O2/SU-8 with a well depth of 20 mih.
Design 1
A standard design of 'active' wells, Design 1, is a square array of 75 mih wells, pitched at
250 m along the X and Y axes. Pre-treatment was applied to Design 1 using by dip-coating an
SU-8 and silicon piece in a pre-treatment solution of 10% pristane (2,6,10,14-
tetramethylpentadecane) in hexane, at a velocity of approximately 1 mm/s.
Lipid bilayers were prepared in the following way. The micro-wells were first filled with
a solution of lipid vesicles in buffer (3.6 g/L of l,2-diphytanoyl-sn-glycero-3-phosphocholine in
a buffer composed of 400 mM KC1, 25 mM Tris in water). An air-solution interface was then
created by slowly retracting the excess lipid solution from the flow cell. The lipid bilayers were
then painted by slowly introducing the solution of lipid in the flow cell (the optical dye
sulforhodamine 101 (green excitation, red emission) was added to the lipid solution at the
concentration of O.Olg/L). The meniscus of the introduced solution effectively paints lipid
bilayers on the micro-wells. The excess lipids were then flushed by a large volume of buffer.
Thereafter, the presence of lipid bilayers was determined by epifluorescence, using the
optical dye introduced to the lipid solution, which was trapped in the wells as the bilayer formed.
A representative image, giving a general overview of the result (without particular detail of the
wells), is shown in Fig 6, in which brighter areas represent the presence of pre-treatment.
As can be seen, the quality of pre-treatment is variable, with some wells not showing the
presence of any pre-treatment at all. Counting a bilayer as present if it covers a micro-well
entirely, standard image processing methods of particle counting can be used to analyse the
epifluorescence images. An average of 68.5% bilayer formation was found, after 3 tests, with a
standard deviation of 2.7%.
To determine the effect of the design parameters on the quality of bilayer formation,
further experiments were conducted.
In the following examples arrays of wells were mounted in flow cell. Pre-treatment (10%
pristane in hexane, IOOmI) was pushed through the array chip at a flow rate of IOOmI/s. The chips
were then dried in one of two methods. (1) By removing the connecting pipe-work and placing
the array chip in a desiccator for 15 minutes under vacuum, at 200 mBar pressure (i.e. below
atmospheric pressure). This allowed the hexane to evaporate leaving behind the pristane in the
location it is deposited. (2) By pushing air through the array chip at a constant, but low, flow
rate for 15 minutes. This allowed the hexane to evaporate at atmospheric pressure, but the
vapour removed which drives the drying process.
Design 2
The design had 75 mih wells, pitched at 250 mih along the X and Y axes. These were
interleaved with the same design off set 125 mih on the X and Y axes, effectively producing a
square array of 75 mih wells, pitched at 177 mih along axes angled at 45° to the X and Y axes.
This design, Design 2, doubles the micro-well density on the SU-8 array compared to Design 1.
Representative images of these results are shown in Figs 7a-f, in which Fig. 7a is an
overview of a desiccator drying experiment (and does not provide particular detail of the wells),
Fig. 7b is close up of a desiccator drying experiment, Fig. 7c is an overview of a pump drying
experiment (and does not provide particular detail of the wells), Fig. 7d is a close up of a pump
drying experiment.
Using the desiccator drying method, as shown in Figs. 7a and 7b, an acceptable pretreatment
distribution was obtained. The arrays when dried in this way do not show any
significant signs of pre-treatment on the surface of the SU-8 (i.e. between the micro-wells).
However, it is noted that the arrays did not seem particularly even in intensity, and the overall
intensity was low.
Using the pump drying method, as shown in Figs. 7c and 7d, the results were clearly
unsatisfactory. Much of the surface was covered in larger pools of pre-treatment and many of
the micro-wells were filled with pre-treatment.
Although this may lead to the conclusion that the pre-treatment drying method is the
most important factor in obtaining a good pre-treatment distribution, it is not the only
consideration. As shown in Figs. 8a and 8b, a sample produced via dip coating the pretreatment
(rather than the painting technique used for Figs. 7a-d), produces a satisfactory but u n
even distribution, even though the surface is clear of pre-treatment. Fig. 8a is an overview of the
example produced via dip coating (and does not provide particular detail of the wells), and Fig.
8b is a close up of the example produced via dip coating.
Design 3
A design having: 75 mh wells, squarely pitched at 125 mih on both X and Y axes, was
used as Design 3 . This effectively represents a grid of 'active' wells as in Design 1, with an
additional array of 'inactive' wells also of 75 mih diameter and squarely pitched at (0, 125 mih),
(125 mp , 125 mih) and (125 m , 0) on the X and Y axes between the 'active' wells. Design 3
increases the array density by a factor of 4 compared with Design 1.
Representative images of these results are shown in Figs 9a-d, in which Fig. 9a is an
overview of a desiccator drying experiment (and does not provide particular detail of the wells),
Fig. 9b is close up of a desiccator drying experiment, Fig. 9c is an overview of a pump drying
experiment (and does not provide particular detail of the wells), Fig. 9d is a close up of a pump
drying experiment. The background brightness running through Figs. 9c and 9d is pre-treatment
deposited on the top surface of the viewing cell and not on the chip surface.
As can be seen, desiccator drying resulted in the surface of the chip being completely
homogeneous with respect to the pre-treatment. There is little, if any, pre-treatment sat on the
SU-8 surface between the micro-wells.
Pump Drying provides an improvement over Design 2 (which has a well density half that
of Design 3). However, this design still leads to significant filling of the micro-wells towards the
front of the array, less so towards the rear of the chip. This is probably due to flow rate
variations over the surface of the chip. Moreover, we can see the pinning effects of the microwells;
in many cases the pre-treatment is pinned on the top SU-8 surface rather than filling the
micro-wells (producing the square looking blobs between wells in Figs. 9c and 9d). This is an
unsatisfactory result.
Design 4
Design 4 utilised wells of different shaped micro-wells, to investigate the effect the well
shape has on the quality of the pre-treatment. Changing the well shape changes the aspect ratio
of the area covered and also probes if any pinning is due to the shape (and symmetry) of the
micro-wells.
Design 4 uses the same pitch as Design 3 (square pitch of 125 mp on both X and Y axes).
However as shown in Fig. 10a, instead of an array of only circular wells (as in Design 4), an
array based on the repeating pattern of one circular well and three square wells (arranged so that
the four wells form a square on the array), was used as Design 4 . Each circular well had a
diameter of 75 mp , whilst the square wells had a side length of 75 m .
In this design, the circular wells can be considered as representing 'active' wells, whilst
the square wells represent 'inactive' wells. Therefore, Design 4 corresponds to Design 3, but
with the shape of the 'inactive' wells changed.
Representative images of these results are shown in Figs 10 b-e, in which Fig. 10b is an
overview of a desiccator drying experiment (and does not provide particular detail of the wells),
Fig. 10c is close up of a desiccator drying experiment, Fig. lOd is an overview of a pump drying
experiment (and does not provide particular detail of the wells), Fig. lOe is a close up of a pump
drying experiment. Once again, the bright background in Figs. lOd and lOe is due to pretreatment
deposited on the view cell not on the chip surface.
As can be seen, desiccator drying provided a very similar result to the "all circular"
equivalent of Design 3 . The shape does not seem to affect the amount of pre-treatment
remaining on the surface. That is, the change in shape does not make the quality of pretreatment
worse.
Indeed, the pump drying experiment indicates the change in shape has a positive effect.
In the pump dried example (Figs. lOd and lOe), the amount of surface remaining pre-treatment is
quite high as in the "all round" counterpart. However, only the square micro-wells have filled.
As a result, the quality of pre-treatment is acceptable.
Design 5
Design 5 (shown in Fig. 11a) removes a large amount of the SU-8 surface. 75 m
circular wells are arranged on a square pitch oft 250 m on both X and Y axes, as in Design 1.
In addition, a 'background' pattern of 20um squares with a 5 m boarder is provided. The use of
a square background pattern, closely spaced, provides an efficient pattern for removing as much
surface material as possible, whilst providing a texture.
Representative images of these results are shown in Figs 11 b-e, in which Fig. 1lb is an
overview of a desiccator drying experiment (and does not provide particular detail of the wells),
Fig. 1l c is close up of a desiccator drying experiment, Fig. 1Id is an overview of a pump drying
experiment (and does not provide particular detail of the wells), Fig. 1l e is a close up of a pump
drying experiment. Once again, the bright background in Figs l i d and 1l e is due to pretreatment
deposited on the view cell not on the chip surface.
As expected in view of the results for Designs 3 and 4, desiccator drying of Design 5
provided a surface that is very uniform and free of excess pre-treatment. The small micro-wells
make it difficult to see the pre-treatment, but it is very uniform over the whole surface. The
variation in background intensity in Fig. 1lb is due to pre-treatment on the view cell, not the chip
surface. Further, the bright area at the bottom right was found to be caused by dust on the
surface, pinning more pre-treatment, but it is notable no filled wells were produced even in this
region.
The pump drying experiment provided an apparently identical result (barring variations
due to the presence of pre-treatment on the view-cell) to the desiccator drying experiment.
We look to the designs that we short-listed, namely the 50-81 and 50-63 (which denotes
the size of the micro-patterned wells and their pitched spacing - in m ) . We know that
desiccator drying methods at this scale work well for both designs, since the 125 mp pitched
micro-patterned wells performs well under these conditions.
Designs 6 and 7
Designs 6 and 7 are also based upon an 'active' array of 75 m circular wells are
arranged on a square pitch oft 250 m on both X and Y axes, as in Design 1. In addition, Design
6 (Fig. 12 a) incorporates a background pattern of 50 m circular 'inactive' wells between the
'active' wells, on a square pitch of 8 1 mp , whilst Design 7 (Fig. 13a) incorporates a background
pattern of 50 m circular 'inactive' wells between the 'active' wells, on a square pitch of 63 m .
As such, in these designs the cross-sectional area of the aperture of the additional (or 'inactive'
or 'flow control') wells is less than the area of the 'active' or 'sensor' wells.
Only pump drying experiments were performed for these designs, as it can be inferred
from the results for Design 3 that desiccator drying will work well.
Representative images of the results for Design 6 are shown in Figs. 12b and 12c, in
which Fig. 12b is an overview of a desiccator drying experiment (and does not provide particular
detail of the wells), and Fig. 12c is close up of a desiccator drying experiment. Representative
images of the results for Design 7 are shown in Figs. 13b and 13c, in which Fig. 13b is an
overview of a desiccator drying experiment (and does not provide particular detail of the wells),
and Fig. 13c is close up of a desiccator drying experiment.
Satisfactory results were obtained from Design 6 . The majority of the surface is uniform,
(there is some variation in the pre-treatment over the surface but this may be more related to the
surface chemistry of the chip), however there are on average only a few micro-wells that are
filled or are non-uniform compared to the majority of the micro-wells for which the pretreatment
forms in a uniform manner..
Good results were obtained from Design 7 . Accounting for the obvious view cell
variations, there do not appear to be any filled micro-wells, and the distribution appears to be
more homogeneous compared to Design 6
The results for the above discussed designs have been quantified, and tabulated in Table
2, by allocating the quality of the pre-treatment distribution a grade. In order to do this, the
homogeneity of the pre-treatment distributions were assessed by image analysis, in order to
measure the rectangularity and perimeter of the pre-treatment in the wells. The rectangularity is
defined as the ratio of the cross-sectional area of pretreatment to the cross-sectional area of a
notional inscribed (non-rotational) rectangle within the well (i.e. the rectangle with the largest
cross-sectional area which can be inscribed within the pre-treatment cross-sectional area). This
ratio is pi/4 for a perfect circular object and unity for a non-rotated rectangle. To do this, the
image being analysed was split into its red, green and blue components. A green fluorescent dye
(a boron-dipyrromethene) was used to highlight the pretreatment and a red fluorescent dye
(sulforhodamine) was used to highlight the buffer under the membrane layer. The gray-scale
image was then threshold filtered just above background level. The duotone image was then
subjected to a shape analysis on each object identified. On this basis the following grades were
defined:
Grade 1 :
• the visible pre-treatment coverage of the surface is lower than 5% of the surface of the
array in the fluidic cell
· the number of filled wells in the array (both 'active' and 'inactive') is smaller than 0.5%
• the homogeneity of the distribution of the pre-treatment annuli in the wells is high, as
quantified by the rectangularity and perimeter being is within ± 20% of the average value. For
example, if the average value of the perimeter is 140 for a 50 m well, then all the perimeters
measured on the 50 m wells needs to be in the interval of 112 m to 168 m .
Grade 2
• 5% < surface coverage by pre-treatment < 15%
• 0.5 % < number of filled wells < 5%
• ± 20% of average < intervals for characteristics of the annuli < ± 40% of average
Grade 3
• surface coverage by pre-treatment > 15%
• number of filled wells > 5%
• intervals for characteristics of the annuli > ± 40% of average
'Additional' X Pitch Y Pitch Drying Grade
Well (mih) ( h ) Method
Diameter
(mih)
Design 2 75 250 250 Vacuum 2
Design 2 75 250 250 Pump 3
Design 3 75 125 125 Vacuum 1
Design 3 75 125 125 Pump 3
Design 4 75 (square) 125 125 Vacuum 1
Design 4 75 (square) 125 125 Pump 2
Design 5 15 (square) 5 5 Vacuum 1
Design 5 15 (square) 5 5 Pump 1
Design 6 50 8 1 8 1 Pump 2
Design 7 50 63 63 Pump 1
Table 2 : Summary of results for Designs 2-7.
As can be seen from Table 2, and the forgoing discussion, vacuum/desiccator drying
provides better quality distributions for similar well geometries than pump/convection drying for
less textured (i.e. having larger, more spaced apart additional wells) surfaces. However, for
highly textured surfaces the drying method does not affect the grade of pre-treatment obtained
(i.e. as shown by Design 5).
It can also be seen that is preferable to have more closely spaced 'additional' wells (e.g.
by comparing Designs 2 and 3), to obtain better quality pre-treatment distribution. Preferably
the additional wells are spaced at 125 mih apart or less, more preferably 100 mih apart or less,
more preferably 8 1 mih or less, more preferably 63 mih or less. Preferably, the pitch of the
additional wells is smaller than the pitch of the array of 'sensor' or 'active' wells.
It is also possible to calculate the number density of wells (wells/micron 2), area density of
wells (well area/ total area), nearest-neighbour distance between wells for the Designs 1 to 3 .
These designs represent designs have only one shape of well is present (both in terms of
geometry and size). These values are quantified in Table 3 .
Well Number Density Well Area Density Well Nearest
(wells/micron2) (-) Neighbour Distance
(microns)
Design 1 1.6xl0 5 0.071 175
Design 2 3.2xl0 5 0.141 102
Design 3 6.4xl0 5 0.283 50
Table 3 : Bulk characteristics of Designs L-3
From the trends in Tables 2 and 3, it is apparent that it is preferable to have a higher
density of wells on the surface to provide a better pre-treatment distribution. Preferably, the
number distribution of wells (whether active or inactive) is at least 3.2xl0 5 wells/micron 2, more
preferably 6.4xl0 5 wells/micron 2. Preferably, the well area density is 0.141 or more, more
preferably 0.283 or more. Preferably the wells are formed so that the distance to the next nearest
well is 102 microns away or less, more preferably 50 microns or less.
It is also contemplated that future apparatuses may reduce further in size, in which case
the 'additional' wells provided in Designs 6 and 7, may actually be used as a continuous array of
active wells. In that case, the Designs would have the characteristics shown in Table 4 .
Table 4 : Bulk characteristics of 'additional' wells of Designs 6 and 7
As such, the number distribution of wells is still more preferably 1.5xl0 4 wells/micron 2
or more, and still more preferably 2.5xl0 4 wells/micron 2 or more. Further the well area density
is still more preferably 0.299 or more, and still more preferably 0.495 or more. Additionally, the
wells are still more preferably formed so that the next nearest well is 31 microns away or less,
and more preferably 13 microns away or less.
It is further apparent from Table 1 that is preferable for the wells, whether they are all
active or not, to be smaller. Preferably the wells are 75 microns in diameter or smaller, more
preferably 50 microns in diameter or smaller.
In practice, the advantage of the present invention may be achieved using arrays
constructed either only partially or entirely of active wells. By controlling the surface energy by
using the additional wells (whether they are ultimately used for sensing or otherwise) an
improved flow of the pre-treatment can be obtained as well as an improvement of the subsequent
pre-treatment distribution. Even in the absence of a pre-treatment step, the improved flow
control gives more uniform flows that can help bilayer formation.
The present invention has been described above with reference to specific embodiments.
It will be understood that the above description does not limit the present invention, which is
defined in the appended claims.

CLAIMS
1. An apparatus for supporting an array of layers of amphiphilic molecules, the apparatus
comprising:
a body,
formed in a surface of the body, an array of sensor wells capable of supporting a layer of
amphiphilic molecules across the sensor wells, the sensor wells each containing an electrode
for connection to an electrical circuit, and
formed in the surface of the body between the sensor wells, flow control wells capable of
smoothing the flow of a fluid across the surface.
2 . An apparatus according to claim 1, wherein the cross-sectional area of a flow control well is
less than the area of a sensor well.
3 . An apparatus according to claim 1 or claim 2, wherein the flow control wells do not contain
electrodes.
4 . An apparatus according to claim 1 or claim 2, wherein the flow control wells each contain an
electrode, the electrodes in the sensor wells being connected to the electrical circuit but the
electrodes in the flow control wells not being connected to the electrical circuit.
5 . An apparatus according to any one of claims 1 to 4, further comprising a cover over the
surface of the body defining a cavity therebetween, and a common electrode arranged in the
cavity for connection to the electrical circuit.
6 . An apparatus according to claim 5, wherein the cover has an internal surface facing the
surface of the body that is roughened to smooth the flow of fluid thereover.
7 . An apparatus according to any one of claims 1 to 6, wherein the array of sensor wells is a
regular array, and the flow control wells consist of a regular array of flow control wells.
8 . An apparatus according to any one of claims 1 to 7, wherein the pitch of at least a portion of
the array of flow control wells is smaller than the pitch of at least a portion of the array of
sensor wells.
9 . An apparatus according to any one of claims 1 to 8, wherein the sensor wells are circular.
Claim 9a An apparatus according to any one of claims 1 to 9 wherein the flow control wells
are square.
10. An apparatus according to any one of claims 1 to 9, wherein the flow control wells are
distributed over a larger area than the sensor wells.
11. An apparatus according to any one of claims 1 to 10, wherein the sensor wells and flow
control wells are arranged such that a pre-treatment of a hydrophobic fluid applied to the
surface of the body would not enter the Cassie-Baxter state.
12. An apparatus according to any one of claims 1 to 11, wherein the sensor wells and flow
control wells are shaped to provide a surface roughness r, defined as the total area of the
surface and wells divided by the projected area of the surface, and a solid surface area
fraction / , defined as the area of the surface between the wells divided by the projected area
of the surface, that meet the requirement in respect of a pre-treatment, that is a fluid capable
of interacting with the amphiphilic molecules, having a contact angle Qthat {{f -\)l{r-
0>))>COS( ) .
13. An apparatus according to any one of claims 1 to12, wherein the wells are formed on the
surface with a number density of 3 .2xl0 5 wells/micron 2 or more, optionally 6.4xl0 5
wells/micron 2 or more, further optionally 1.5xl0 4 wells/micron 2 or more, and still further
optionally 2.5xl0 4 wells/micron 2 or more.
14. An apparatus according to any one of claims 1 to 13, wherein further comprising a pretreatment,
that is a fluid capable of interacting with the amphiphilic molecules, applied to the
sensor wells.
15. A method of preparing an apparatus for forming an array of layers of amphiphilic molecules,
the method comprising:
providing an apparatus according to any one of claims 1 to 13;
delivering across the surface of the body a pre-treatment of a hydrophobic fluid.
16. A method according to claim 15, wherein the pre-treatment is delivered in a solvent, the
method further comprising drying the surface of the body to remove the solvent.
17. A method according to claim 16, wherein said step of drying the surface of the body to
remove the solvent is performed under a pressure below atmospheric pressure.
18. A method according to any one of claims 15 to 17, wherein the method is performed so that
each of the following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 15% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values.
19. .A method according to any one of claims 15 to 18, wherein the method is performed so that
each of the following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 5% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 0.5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 20% of the average values.
20. A method of forming an array of layers of amphiphilic molecules, the method comprising:
preparing an apparatus by a method according to any one of claims 15 to 19; and
flowing a fluid containing amphiphilic molecules across the surface of the body to
form layers of amphiphilic molecules across at least some of the array of sensor wells.
21. A method of forming an array of layers of amphiphilic molecules, the method comprising:
providing an apparatus according to any one of claims 1 to 13; and
flowing a fluid containing amphiphilic molecules across the surface of the body to form
layers of amphiphilic molecules across at least some of the array of sensor wells.
22. An apparatus for supporting an array of layers of amphiphilic molecules, the apparatus
comprising:
a body; and
formed in a surface of the body, an array of wells, at least some of which are sensor wells
capable of supporting a layer of amphiphilic molecules across the sensor wells after
application to the sensor wells of a pre-treatment that is a fluid capable of interacting with the
amphiphilic molecules, the sensor wells each containing an electrode for connection to an
electrical circuit,
wherein the wells have a number density of 3 .2xl0 5 wells/micron 2 or more, optionally
6.4xl0 5 wells/micron 2 or more, further optionally 1.5xl0 4 wells/micron 2 or more, and still
further optionally 2.5xl0 4 wells/micron 2 or more.
23. An apparatus according to claim 22, wherein all the wells are sensor wells.
24. An apparatus according to claim 22, wherein some of the wells are sensor wells, and the
remainder of the wells are flow control wells, formed in the surface of the body between the
sensor wells.
25. An apparatus according to claim 24, wherein the area of a flow control well is less than the
area of a sensor well.
26. An apparatus according to claim 24 or claim 25, wherein the flow control wells do not
contain electrodes.
27. An apparatus according to claim 24 or claim 25, wherein the flow control wells each contain
an electrode, the electrodes in the sensor wells being connected to the electrical circuit but
the electrodes in the flow control wells not being connected to the electrical circuit.
28. An apparatus according to any one of claims 24 to 27, wherein the array of sensor wells is a
regular array, and the flow control wells consist of a regular array of flow control wells.
29. An apparatus according to any one of claims 24 to 28, wherein a pitch of the array of flow
control wells is smaller than a pitch of the array of sensor wells.
30. An apparatus according to any one of claims 24 to 29, wherein the sensor wells are circular,
and the flow control wells are square.
31. An apparatus according to any one of claims 24 to 30, wherein the flow control wells are
distributed over a larger area than the sensor wells.
32. An apparatus according to any one of claims 24 to3 1, further comprising a cover over the
surface of the body defining a cavity therebetween, and a common electrode arranged in the
cavity for connection to the electrical circuit.
33. An apparatus according to claim 32, wherein the cover has an internal surface facing the
surface of the body that is roughened to smooth the flow of fluid thereover.
34. An apparatus according to any one of claims 22 to 33, wherein the wells have an area density
of 0.141 or more.
35. An apparatus according to any one of claims 22 to 34, wherein the wells are arranged such
that a pre-treatment, that is a fluid capable of interacting with the amphiphilic molecules, on
the surface does not enter the Cassie-Baxter state.
36. An apparatus according to any one of claims 22 to 35, wherein the sensor wells and flow
control wells are shaped to provide a surface roughness r, defined as the total area of the
surface and wells divided by the projected area of the surface, and a solid surface area
fraction / , defined as the area of the surface between the wells divided by the projected area
of the surface, that meet the requirement in respect of a pre-treatment, that is a fluid capable
of interacting with the amphiphilic molecules, having a contact angle Qthat {{f -\)l{r-
0>))>COS( ) .
37. An apparatus according to any one of claims 22 to 34, wherein further comprising a pretreatment,
that is a fluid capable of interacting with the amphiphilic molecules, applied to the
sensor wells.
38. A method of preparing an apparatus for forming an array of layers of amphiphilic molecules,
the method comprising:
providing an apparatus according to any one of claims 22 to 36;
delivering across the surface of the body a pre-treatment that is a fluid capable of
interacting with the amphiphilic molecules to apply the pre-treatment to the wells.
39. A method according to claim 38, wherein the pre-treatment is delivered in a solvent, the
method further comprising drying the surface of the body to remove the solvent.
40. A method according to claim 39, wherein said step of drying the surface of the body to
remove the solvent is performed under a pressure below atmospheric pressure.
41. A method according to any one of claims 38 to 40, wherein the method is performed so that
each of the following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 15% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values.
42. A method according to claim 41, wherein the method is performed so that each of the
following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 5% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 0.5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 20% of the average values.
43. A method of forming an array of layers of amphiphilic molecules, the method comprising:
preparing an apparatus by a method according to any one of claims 38 to 42; and
flowing a fluid containing amphiphilic molecules across the surface of the body to form
layers of amphiphilic molecules across at least some of the array of sensor wells.
44. A method of preparing an apparatus for forming an array of layers of amphiphilic molecules,
the method comprising:
providing an apparatus comprising a body, and, formed in a surface of the body, an array
of wells, at least some of which are sensor wells capable of supporting a layer of amphiphilic
molecules across the sensor wells after application to the sensor wells of a pre-treatment that
is a fluid capable of interacting with the amphiphilic molecules, the sensor wells each
containing an electrode for connection to an electrical circuit, and
delivering across the surface of the body a pre-treatment, that is a fluid capable of
interacting with the amphiphilic molecules, in a solvent to apply the pre-treatment to the
wells; and
drying the surface of the body to remove the solvent under a pressure below atmospheric
pressure.
45. A method according to claim 44, wherein the apparatus is an apparatus according to any one
of claims 22 to 36.
46. A method according to claim 44 or claim 45, wherein the method being performed so that
each of the following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 15% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values.
47. A method according to claim 46, wherein the method is performed so that each of the
following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 5% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 0.5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 20% of the average values.
48. A method of forming an array of layers of amphiphilic molecules, the method comprising:
preparing an apparatus by a method according to any one of claims 44 to 46; and
flowing a fluid containing amphiphilic molecules across the surface of the body to form
layers of amphiphilic molecules across at least some of the array of sensor wells.
49. A method of preparing an apparatus for forming an array of layers of amphiphilic molecules,
the method comprising:
providing an apparatus comprising a body, and, formed in a surface of the body, an array
of wells, at least some of which are sensor wells capable of supporting a layer of amphiphilic
molecules across the sensor wells after application to the sensor wells of a pre-treatment that
is a fluid capable of interacting with the amphiphilic molecules, the sensor wells each
containing an electrode for connection to an electrical circuit, and
delivering to the body a pre-treatment that is a fluid capable of interacting with the
amphiphilic molecules,
the method being performed so that each of the following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 15% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values.
50. A method according to claim 49, wherein the method is performed so that each of the
following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 5% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 0.5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 20% of the average values.
51. A method according to claim 49 or 50, wherein the pre-treatment is applied to the body in a
solvent, and the method further comprises drying the surface of the body to remove the
solvent, the method being performed so that said conditions are met after said drying.
52. A method of forming an array of layers of amphiphilic molecules, the method comprising:
preparing an apparatus according to any one of claims 49 to 51; and
flowing a fluid containing amphiphilic molecules across the surface of the body to form
layers of amphiphilic molecules across at least some of the array of sensor wells.
53. An apparatus for forming an array of layers of amphiphilic molecules, the apparatus
comprising:
a body; and
formed in a surface of the body, an array of wells, at least some of which are sensor wells
capable of supporting a layer of amphiphilic molecules across the sensor wells after application
to the sensor wells of a pre-treatment that is a fluid capable of interacting with the amphiphilic
molecules, the sensor wells each containing an electrode for connection to an electrical circuit,
the array of wells being arranged such that after delivery to the body of a pre-treatment that is a
fluid capable of interacting with the amphiphilic molecules, each of the following conditions is
met:
the visible coverage of the surface by the pre-treatment is less than 15% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 40% of the average values.
54. An apparatus according to claim 53, wherein the array of wells is arranged such that after
delivery to the body of a pre-treatment that is a fluid capable of interacting with the
amphiphilic molecules, each of the following conditions is met:
the visible coverage of the surface by the pre-treatment is less than 5% of the area in
which the array of sensor wells is located;
the proportion of sensor wells that are filled is less than 0.5%; and
the values of rectangularity and the perimeter of all the annuli of pre-treatment around the
respective sensor wells falls within a 20% of the average values.
55. An apparatus according to claim 54, wherein further comprising a pre-treatment, that is a
fluid capable of interacting with the amphiphilic molecules, applied to the sensor wells.