Field of the Invention
This invention relates to mass spectrometry, and in particular
to the use of mass spectrometry in conjunction with liquid chromatography or capillary
electrophoresis. The invention particularly relates to a system and method that
is implemented in a microengineered configuration.
Electrospray is a common method of soft ionisation in biochemical
mass spectrometry (MS), since it allows the analysis of fluid samples pre-separated
by liquid chromatography (LC), the ionization of complex molecules without fragmentation,
and a reduction in the mass-to-charge ratio of heavy molecules by multiple charging
[Gaskell 1997; Abian 1999]. It may be used in a similar way with fluid samples pre-separated
by other methods such as capillary electrophoresis (CE).
The principle is simple. A voltage is applied between an
electrode typically consisting of a diaphram containing an orifice and a capillary
needle containing the analyte. Liquid is extracted from the tip and drawn into a
Taylor cone, from which large charged droplets are emitted. The droplets are accelerated
to supersonic speed, evaporating as they travel. Coulomb repulsion of the charges
in the shrinking droplet results in fragmentation to ions when the Rayleigh stability
limit is reached. The resulting ions can be multiply charged.
An electrospray mass spectrometer system contains a number
of key elements:
- An electrospray ionisation source capable of interfacing to an LC or CE system
- An interface to couple ions (in preference to molecules) into a vacuum chamber
- An alignment and/or observation system capable of maximising the coupling
- A mass filter and detector
Conventionally, the spray is passed from atmospheric pressure
via a chamber held at an intermediate pressure. Several vacuum interfaces that use
differential pumping to match flow rates to achievable pressures have been developed
[Duffin 1992]. The ion optics normally consist of input and output orifices such
as capillaries, capillary arrays and skimmer electrodes, and occasionally also a
quadrupole lens operating as an ion guide in all-pass mode. These components are
used to maximise the ratio of coupled ions to neutrals, which would otherwise swamp
Various methods are used to promote a well-dispersed spray
of small droplets and hence a concentrated flow of analyte ions. Solvent can be
preferentially driven off, by direct heating [Lee 1992]. Advantages may be obtained
by the use of a sheath gas flow [Huggins 1993], and nebulisation may be enhanced
by ultrasound [Hirabayashi 1998].
Alignment in electrospray is not critical, and the spray
may simply be directed towards the MS input. Alternatively, an off-axis spray direction
may be used to promote the separation of neutrals. Co-axial lenses mounted directly
on the capillary have been developed to focus the spray [
]; however, there are limits to the electrode complexity that can be achieved
using such simple mechanical systems.
In a conventional electrospray system, with capillaries
of ≈100 µm internal diameter, flow rates are of the order of 1 µl
min-1, and extraction voltages lie in the range 2.5 kV - 4 kV. Flow rates
and voltages are considerably reduced in so-called "nanospray systems", based on
capillaries having internal diameters ranging down to ≈10 µm [Wilm 1996].
Such capillaries are relatively easy to fabricate, and are available with a range
of diameters and frits. Decreasing the capillary diameter and lowering the flow
rate also tends to create ions with higher mass-to-charge ratio, extending the applicability
further towards biomolecules.
Because of the reduced size of the spray cone, alignment
of a nanospray source is more critical. Operation typically involves mounting the
source on a micropositioner and using a video camera to observe the spray entering
the vacuum inlet of an atmospheric pressure ionisation (API) mass spectrometer.
Sources are sold customised for most popular brands of mass spectrometer. However,
such systems are large, complex and costly.
To reduce costs, a variety of attempts have been made to
integrate some of the components of nanospray ionisation sources. Ramsey and Ramsey
 showed that a spray could be drawn from the edge of a glass chip containing
an etched capillary. Since then, integrated capillaries with in-plane flow have
been demonstrated in many materials, especially plastics [Licklider 2000; Svedberg
2003]. In some cases, the fluid has been extracted from a slot rather than a channel
[Le Gac 2003]; in others, from a shaped surface [Kameoka 2002]. Devices have also
been formed in one-dimensional arrays. Geometries in which the flow is passed perpendicular
to the surface of the chip have also been demonstrated, often by deep reactive ion
etching of silicon [Schultz 2000; Griss 2002]. Such devices may be formed into two-dimensional
Almost exclusively, the advances above consist of attempts
to integrate system subcomponents leading up to the ion emitter. They concentrate
on the fluidic part of the system, ignoring the problems of separating ions from
neutrals, and of aligning the ion spray to the inlet to the vacuum system. As a
result, they are not suitable for a low cost nanospray system, because accurate
alignment still requires expensive positioning devices.
There is therefore a need to provide a low cost nanospray
The invention addresses these and other problems by providing
a solution to the problems of alignment and electrode mounting in a low-cost nanospray
source by using microelectromechanical systems technology to form appropriate mechanical
alignment and conducting electrode features on insulating plastic substrates in
an integrated manner. The approach also allows integration of features for fluid
drainage, spray heating and sheath gas flow.
This invention provides a method of aligning a nanospray
capillary needle, a set of electrodes, and the capillary input to an API mass spectrometer.
The electrode system is formed using microelectromechanical systems technology,
as an assembly of two separate chips. Each chip is formed on an insulating plastic
substrate. The first chip carries mechanical alignment features for the capillary
electrospray needle and the API mass spectrometer input, together with a set of
partial electrodes. The second chip carries a set of partial electrodes. The complete
electrode system is formed when the chips are assembled in a stacked configuration,
and consists of an einzel lens capable of initiating a Taylor cone and separating
ions from neutrals by focusing.
Accordingly, the invention provides a system according
to claim 1 with advantageous embodiments provided in the dependent claims thereto.
The invention also provides a method of fabricating such a system as detailed in
the main independent method claim.
These and other features will be better understood with
reference to the following drawings.
Brief Description of the Drawing
Detailed description of the Drawings
- Figure 1 shows in schematic form a microengineered nanospray system aligning
a nanospray needle with the capillary input to an atmospheric pressure ionisation
mass spectrometer according to an embodiment of the present invention.
- Figure 2 shows construction of a microengineered nanospray system as a stacked
assembly of two chips according to an embodiment of the present invention.
- Figure 3 is a process flow for construction of a microengineered nanospray chip
according to an embodiment of the present invention.
- Figure 4a shows the layout of a lower and Figure 4b the latout of an upper substrate
of a microenginered nanospray chip according to an embodiment of the present invention.
- Figure 5 shows an assembly of a microengineered nanospray chip according to
an embodiment of the present invention.
- Figure 6 shows electrostatic operation of a microengineered nanospray chip according
to an embodiment of the present invention.
- Figure 7 shows operation of the sheath gas inlet of a microenginered electrospray
chip according to an embodiment of the present invention.
- Figure 8 shows thermal operation of a microengineered electrospray chip according
to an embodiment of the present invention.
- Figure 9 shows electrode configurations realisable using a stacked electrode
assembly with Figure 9a) being a closed pupil arrangement, Figure 9b) a horizontally
split pupil, Figure 9c) a vertically split pupil and Figure 9d) a quadrant pupil
The invention will now be described with reference to exemplary
embodiments as provided in Figures 1 to 9.
The present inventor has realised that the benefit of MEMS
structures can be extended to nanospray applications. In MEMS, widely used methods
of lithographic patterning, oxidation and metallisation are combined with specialised
techniques such as anisotropic wet chemical etching [Bean 1978] and deep reactive
ion etching [Hynes 1999] to form three-dimensional features in crystalline semiconductors
such as silicon. UV exposure of specialised photosensitive polymers such as SU-8
may be used to form three-dimensional features in plastics [Lorenz 1997]. These
methods may be used to combine insulating substrates, alignment features and conducting
electrodes. The present inventor has realised that at least potentially, they may
therefore form an integrated nanospray ionisation source at low cost.
However, further difficulties remain with the realisation
that MEMS technology could be used to provide nanospray devices. The device must
typically operate with high voltages, in a wet environment, so that electrical isolation
and drainage are both required. The substrate material most commonly used in MEMS,
silicon, is therefore not appropriate; however, other insulating materials such
as glasses are difficult to micromachine. To obtain a stable spray, an electrode
containing an axially aligned orifice is typically required. To obtain efficient
ion separation from neutrals, electrostatic deflection or focusing is required.
For focusing, further electrodes containing aligned orifices are needed. If the
ion path is itself in the plane of a substrate, such orifices are extremely difficult
to form by in plane patterning alone. Finally, it is desirable to integrate features
capable of providing a sheath gas around the spray, of promoting nebulisation, and
of preferentially evaporating solvent. For these and other reasons there has heretofore
not been possible an integrated MEMS nanospray system. However, as will be understood
from a review of Figures 1 to 9, the present inventor has addressed these and other
Figure 1 illustrates the concept of a microengineered nanospray
electrode system. A mass spectrometer 101 is provided in a high-vacuum enclosure
102 pumped (for example) by a turbomolecular pump 103. Ions are channelled into
this chamber via a further chamber 104 held at an intermediate pressure and pumped
(again, for example) by a rotary pump 105. The inlet to the vacuum system is assumed
to be a capillary 106. The exact configuration of these components is not, it will
be appreciated, important, apart from the input capillary. For example, the filter
element of the mass spectrometer could be an ion trap, a quadrupole, a magnetic
sector, a crossed-field or a time of flight device. Equally, the intermediate vacuum
chamber could contain a range of components including further capillaries and skimmer
The overall input to the system is provided by a nanospray
capillary 107. Alignment between the nanospray capillary 107 and the capillary input
to the mass spectrometer 106 is provided by a microengineered chip 108.. The chip
contains a first set of mechanical alignment features 109 for the nanospray capillary
and a second set of alignment features 110 for the capillary input to the mass spectrometer.
The chip also contains a set of electrodes 111 set up perpendicular to the ion path,
which may (for example, but not exclusively) consist of diaphragm electrodes. Other
features may be integrated on the chip, including holes for drainage and gas inlet.
Figure 2 illustrates the main features of the chip 108.
The chip is constructed from two separate substrates, each carrying microengineered
features, which are arranged in a stacked assembly. The first substrate consists
of a base 201 formed in insulating material and carrying a mechanical alignment
feature for the nanospray capillary corresponding to the feature 109 in Figure 1,
which may (for example, but not exclusively) consist of a groove 202 etched into
a conducting or semiconducting block 203. This substrate also carries an alignment
feature for the capillary input to the mass spectrometer corresponding to the feature
110 in Figure 1, which may again for example consist of a further groove 204 etched
into a block of similar material 205. This substrate also carries a set of electrodes
corresponding to part of the features 111 in Figure 1 and consisting of grooves
206 etched into upright plates of similar material 207.
The second substrate again consists of a base 208 formed
in insulating material, and carrying a further set of electrodes corresponding to
a further part of the features 111 in Figure 1 and consisting of grooves 209 etched
into upright plates of conducting or semiconducting material 210. When the two substrates
are stacked together, the partial electrode sets combine to form complete diaphragm
electrodes with closed pupils 211.
Using three such electrodes, a so-called 'einzel' or unipotential
electrostatic lens is formed. This type of lens allows focusing of ions passing
axially through the stack of electrodes in a simple and controlled manner, and hence
allows the ion spray to be focused onto the capillary input to the mass spectrometer
to present a concentrated stream of analyte ions.
It will be appreciated that the alignment grooves 202 and
204, and the electrode grooves 206 and 209, may all be defined by similar photolithographic
processes, and may therefore be registered together. This aspect provides a solution
to the first problem identified above in the Background to the Invention section,
of constructing an accurately aligned set of mechanical features and electrodes.
It will also be appreciated that the use of an insulating substrate that may be
patterned with drain holes provides a solution to the problem of maintaining high
voltages in a wet environment. Finally it will be appreciated that a stacked combination
of partial electrodes provides a solution to the problem of forming diaphragm electrodes
arranged normal to a substrate.
It will be appreciated by those skilled in the art that
a variety of materials and processes and may be used to realise structures similar
to Figure 2. Figure 3 shows a process, which is intended to be exemplary rather
than exclusive. The materials used are low cost, and only three lithographic steps
are required. The process is based on crystalline silicon substrates on which plastic
virtual substrates are subsequently formed. The individual process steps are indicated
by a set of evolving wafer cross-sections containing typical features.
In step 1, a (100)-oriented silicon substrate 301 is first
oxidised to form a SiO2 layer 302 on both sides. The SiO2
is patterned and etched to form a channel-shaped opening 303, by (for example) photolithography
and reactive ion etching. In step 2, the underlying silicon substrate is anisotropically
etched down (111) crystal planes to form a V-shaped groove 304. Commonly an etchant
consisting of potassium hydroxide (KOH), water and isopropanol (IPA) may be used
for this purpose. This step defines all capillary-mounting grooves and electrode
pupils. The front side oxide is removed, and the wafer is turned over.
In step 3, the wafer is spin coated with a thick layer
of the epoxy-based photoresist
. This resist may be coated and exposed in layers of at least 0.5 mm thickness,
has excellent adhesion, and is extremely rugged after curing, allowing it to be
used as a virtual substrate material after processing. The resist is lithographically
patterned to form a dicing groove 306 around each die, together with any drain holes
307 and gas inlets.
In step 4, the front side of the wafer is metallised to
increase conductivity, typically with an adhesion layer of Cr metal and a further
thicker layer of Au 308. In step 5, the front side of the wafer is coated in a photoresist
309. Since the wafer is non-planar, an electrodeposited resist is used in preference
to spin-coated resist for this step. The resist is patterned to define the outlines
of all electrode and alignment blocks 310, and the pattern is transferred through
the metal. In step 6, the pattern is transferred through the silicon wafer by deep
reactive ion etching, to form deep separation features 311 between elements. The
photoresist is then removed, and individual dies are separated in step 7.
In step 8, two dies are stacked together to form a complete
nanospray chip, by soldering or bonding the metal layers 312 together. Alternatively,
a conducting epoxy may be used for this step. The chip is mounted on a carrier circuit
board, and wirebond connections 313 are made to appropriate features on the lower
It will be appreciated by those skilled in the art that
a first alternative process is offered by forming the conducting alignment and electrode
elements by electroplating a metal inside a mould, which may itself be formed by
a sequence of patterning and etching steps. However, this alternative requires the
separate formation of a mould, which is a laborious process.
It will also be appreciated by those skilled in the art
that a second alternative process is offered by forming the alignment and electrode
elements by sawing or otherwise eroding a conducting layer attached to an insulating
substrate. The substrate bases may be also defined by sawing or by erosion, and
the grooves may be formed, by partial sawing. However, this alternative offers less
flexibility in the range of structures that may be created.
It will also be appreciated by those skilled in the art
that a third alternative process is offered by forming the substrate bases from
glass, which may be patterned by sawing or (in the case of a photosensitive glass)
by photopatterning. However, these alternatives again offer less flexibility in
the range of structure that may be created. It will be appreciated that regardless
of their shortcomings that each of the mentioned alternatives may be considered
useful in the context of the present invention for specific applications.
Figure 4 shows the layout of individual substrates that
can be realised using the process of Figure 3. The larger plastic substrate-base
401 carries a mounting block 402 for the nanospray capillary, formed in etched,
metallised silicon and having an etched alignment groove 403. The substrate carries
a similar mounting block 404 for the mass spectrometer input capillary, with a similar
etched alignment groove 405, and a set of partial electrodes 406 with etched grooves
407. The electrodes are widened at their extremities to assist in the stacked assembly
and to allow bonding. A large hole 408 through the plastic substrate-base provides
a drain, and a smaller hole 409 provides a channel for sheath gas to flow into an
etched plenum chamber 410. The smaller plastic substrate-base 411 carries a further
set of partial electrodes 412 and further features 413 defining the sheath gas plenum.
Figure 5 shows assembly. The smaller substrate 501 is inverted,
aligned on top of the larger substrate 502, and the electrodes are bonded together.
The device is mounted on an external printed circuit board, and wirebond connections
503 are attached to the alignment features and electrodes. The chip is aligned and
connected electrically to the input capillary 504 of the mass spectrometer, and
the nanospray capillary 505 is inserted into its input alignment feature and connected
electrically. A stop may be provided on each capillary to ensure that it may only
be inserted into its alignment groove for a fixed distance.
Figure 6 shows electrostatic operation of the device. The
capillary input to the mass spectrometer and its alignment feature 601 both are
assumed to be at ground potential. Assuming that the nanospray capillary contains
a conducting contact, a large DC voltage V1 is applied to the nanospray
capillary via its associated mount 602. Alternatively the voltage may be applied
via a wire passing into the capillary. An intermediate voltage V2 is
applied to the outer electrodes 603, 604 of the lens element and a further voltage
V3 to the centre element 605. The spray 606 is emitted from a Taylor
cone created at the exit of the nanospray capillary due to the potential difference
V1 ― V2. The ion stream is focused onto the capillary
input to the mass spectrometer 607 due to the action of the focus voltage V3.
Figure 7 shows operation of the sheath gas inlet. Sheath
gas is passed through the lower substrate-base 701 of the assembly via an inlet
hole 702. The gas flows into a plenum 703 formed in the nanospray capillary mount
704. The gas leaks from the plenum around the capillary, because it does not fully
seal the orifice formed by the grooves in the upper and lower nanospray capillary
mount. However, the natural taper of the capillary 705 ensures that the majority
of the leakage takes place in a forward axial direction 706, forming a sheath around
Figure 8 shows a mode of thermal operation. A current I
is passed through one or more of the electrodes 801 to provide local heating, which
may preferentially evaporate more volatile components in the spray such as a carrier
solvent, thus enriching the analyte ion stream.
Figures 9a-9d shows different possible electrode cross
sections. In the simplest realisation (Figure 9a), the assembly of two plates 901
and 902 with grooves formed by anisotropic wet chemical etching will create electrodes
with a diamond-shaped pupil 903. The edges of the pupil will be defined by the (111)
crystal plane angle &thgr; = cos-1(1/√3) = 54.73° of silicon.
The size of the pupils may be controlled, by varying the width of the initial etched
groove either continually or in discrete steps along the axis. It will be appreciated
by those skilled in the art that other fabrication methods such as deep reactive
ion etching may be used to form U-shaped alignment grooves and electrode grooves,
which have greater inherent symmetry.
It will also be appreciated by those skilled in the art
that the electrodes may be segmented horizontally using additional spacing 904 as
shown in Figure 9b, or segmented vertically using additional etching 905 as shown
in Figure 9c. Both methods of segmentation may be combined as shown in Figure 9d.
Segmented electrodes of this type may be used to provide one- or two-axis electrostatic
deflection in addition to focusing. These additional degrees of freedom offer the
potential to improve the separation of ions from neutrals, for example by inserting
a bend or a dog-leg into the ion path that neutrals cannot follow.
It will also be appreciated that the ability to provide
transverse electrostatic forces using segmented electrodes allows the spray to be
deflected in a time-varying manner. If the spray is oscillated using a sinoidally
varying lateral force, a periodic perturbation may be induced in the spray flow.
If the spatial frequency of this perturbation is chosen to coincide with the spatial
frequency of Rayleigh instability in the flow pattern, the flow will be encouraged
to fragment into droplets, thus promoting nebulisation.
What has been described herein is a microengineered nanospray
device. While advantageous embodiments have been described it will be appreciated
that certain integers and components are used to illustrate exemplary embodiments
and it is not intended to limit the invention in any way except as may be deemed
necessary in the light of the appended claims. Furthermore where the invention is
described with reference to specific figures it will be appreciated that components
or features of one figure can be freely interchanged with those of other figures
without departing from the scope of the invention.
While the reference to the miniature nature of the device
of the present invention has been made with reference to MEMS technology it will
be appreciated that within the context of the present invention that the term MEMS
is intended to encompass the terms microengineered or microengineering and is intended
to define the fabrication of three dimensional structures and devices with dimensions
in the order of microns. It combines the technologies of microelectronics and micromachining.
Microelectronics allows the fabrication of integrated circuits from silicon wafers
whereas micromachining is the production of three-dimensional structures, primarily
from silicon wafers. This may be achieved by removal of material from the wafer
or addition of material on or in the wafer. The attractions of microengineering
may be summarised as batch fabrication of devices leading to reduced production
costs, miniaturisation resulting in materials savings, miniaturisation resulting
in faster response times and reduced device invasiveness. Wide varieties of techniques
exist for the microengineering of wafers, and will be well known to the person skilled
in the art. The techniques may be divided into those related to the removal of material
and those pertaining to the deposition or addition of material to the wafer. Examples
of the former include:
- Wet chemical etching (anisotropic and isotropic)
- Electrochemical or photo assisted electrochemical etching
- Dry plasma or reactive ion etching
- Ion beam milling
Whereas examples of the latter include:
- Thick film deposition
- Chemical vapour deposition (CVD)
These techniques can be combined with wafer bonding to
produce complex three-dimensional, examples of which are the interface devices provided
by the present invention.
The words comprises/comprising when used in this specification
are to specify the presence of stated features, integers, steps or components but
does not preclude the presence or addition of one or more other features, integers
, steps, components or groups thereof.
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