MINIATURISATION FOR CHEMISTRY, BIOLOGY & BIOENGINEERING
Development of a micro-fluidic manifold for copper monitoring
utilising chemiluminescence detection
Éadaoin Tyrrell,a Ceri Gibson,b Brian D. MacCraith,b David Gray,b Pat Byrne,b Nigel
Kent,b Conor Burkeb and Brett Paull*a
a National Centre for Sensor Research, School of Chemical Sciences, Dublin City University,
Glasnevin, Dublin 9, Ireland. E-mail: Brett.Paull@dcu.ie; Fax: 00353 (0)1 7005503;
Tel: 00353 (0)1 7005060
b National Centre for Sensor Research, School of Physical Sciences, Dublin City University,
Glasnevin, Dublin 9, Ireland
Received 16th January 2004, Accepted 31st March 2004
First published as an Advance Article on the web 26th April 2004
The progressive development of a micro-fluidic manifold for the chemiluminescent detection of copper in water samples,
based on the measurement of light emitted from the Cu(II) catalysed oxidation of 1,10-phenanthroline by hydrogen
peroxide, is reported. Micro-fluidic manifolds were designed and manufactured from polymethylmethacrylate (PMMA)
using three micro-fabrication techniques, namely hot embossing, laser ablation and direct micro-milling. The final laser
ablated design incorporated a reagent mixing channel of dimensions 7.3 cm in length and 250 3 250 mm in width and
depth (triangular cross section), and a detection channel of 2.1 cm in length and 250 3 250 mm in width and depth (total
approx. volume of between 16 to 22 mL). Optimised reagents conditions were found to be 0.07 mM 1,10-phenanthroline,
containing 0.10 mM cetyltrimethylammonium bromide and 0.075 M sodium hydroxide (reagent 1 delivered at 0.025 mL
min21) and 5% hydrogen peroxide (reagent 2 delivered at 0.025 mL min21). The sample stream was mixed with reagent
1 in the mixing channel and subsequently mixed with reagent 2 at the start of the detection channel. The laser ablated
manifold was found to give a linear response (R2 = 0.998) over the concentration ranges 0–150 mg L21 and be
reproducible (% RSD = 3.4 for five repeat injections of a 75 mg L21 std). Detection limits for Cu(II) were found to be
20 mg L21. Selectivity was investigated using a copper selective mini-chelating column, which showed common cations
found in drinking waters did not cause interference with the detection of Cu(II). Finally the optimised system was
successfully used for trace Cu(II) determinations in a standard reference freshwater sample (SRM 1640).
DOI: 10.1039/b400805g
Introduction
384
The determination of trace elements in environmental samples
requires sensitive and selective analytical techniques. For measurements of trace concentrations of metals in various matrices there are
a number of complex instrumental methods widely used, including
graphite furnace atomic absorption spectroscopy (GF-AAS),
inductively coupled plasma techniques with atomic emission
spectroscopy (ICP-AES) or mass spectrometry (ICP-MS).1 However, there are also many sensitive simpler alternatives to these
complex techniques. The use of chemiluminescence for the
determination of dissolved trace metals has become a popular
choice over recent years as it requires only simple inexpensive
instrumentation compared to the above methods and is often very
sensitive and linear over a wide dynamic range. In addition,
chemiluminescent techniques can be highly selective for certain
species.2 Chemiluminescence is commonly used in flow injection
analysis (FIA) where it delivers low limits of detection, high
precision and fast sample throughput.
Cu(II) is an important element for the metabolism of many living
organisms, but, as with many other metals, at high concentrations it
is toxic. Cu(II) compounds are commonly used in agriculture, for
preservatives and for water treatment. The Environmental Protection Agency (EPA) has stated that drinking water should not
contain more than 1.3 mg L21 of Cu(II),3 with levels above this
producing astringent tastes. The recommended daily allowance
(RDA) for adults is 1.1 mg per day.4 Cu(II) can be determined
sensitively using chemiluminescence. A number of different
chemiluminescent methods have been proposed for the determination of Cu(II) in different sample matrices. Yamada and Suzuki
determined trace amounts of Cu(II) using a chemiluminescent
reaction based on a flavin mononucleotide–hydrogen peroxide–
phosphate buffer system.5 A second method, developed by Yan and
Worsfold, determined Cu(II) by its catalytic effect upon the reaction
Lab Chip, 2004, 4, 384–390
between luminol (5-amino-2,3-dihydrophtalazine-1,4-dione) and
hydrogen peroxide.6 More recently, a method based on the
quenching effect of Cu(II) on the chemiluminescent reaction of
dichlorofluorescein with hydrogen peroxide was developed by
Safavi and Baezzat, and subsequently applied to the determination
of Cu(II) in blood samples.7 Some methods have included an
additional pre-treatment step prior to detection to improve
selectivity. One such example used a micro-column of immobilised
8-hydroxyquinoline (8-HQ) for analyte preconcentration and
removal of matrix interferences in seawater analysis,8 although
later work by Zamzow et al.9 showed how the chemiluminescent
reaction involved could, under certain conditions, be applied
directly to the analysis of seawater without the use of such a
column. The reaction involved was a well established reaction for
Cu(II) determinations, based upon the formation of a complex
between Cu(II) and 1,10-phenanthroline.8–12 The chemiluminescent reaction, which emits between 445–450 nm, results from the
oxidative destruction of the Cu(II)–1,10-phenanthroline complex
by hydrogen peroxide at an alkaline pH, with increased sensitivity
obtained through the addition of a cationic surfactant.10
Chemiluminescence is a promising method of detection for
micro-fluidic analytical systems due to its high sensitivity and the
simplicity of the measurement technique. The combination of
chemiluminescence detection, with its simple instrumentation
requirements, and micro-fluidic systems, with low reagent consumption and portability, is proving very successful, with a number
of chemical and biological flow sensor systems having already been
developed.13–15 This promising combination is aided by recent
progress in the development of fabrication methods for plastic
micro-fluidic manifolds made from polymers such as poly(methylmethacrylate) (PMMA) or poly(dimethylsiloxane)
(PDMS).16
In the work described here, the performance of PMMA microfluidic manifolds fabricated using a variety of micro-fabrication
This journal is © The Royal Society of Chemistry 2004
techniques has been investigated. In combination with chemiluminescence detection, these manifolds have been applied to the
determination and monitoring of Cu(II) in water samples. The aim
of the study was to develop an analytical system, capable of low mg
L21 determinations of Cu(II) in water samples, and which could be
applied to ‘on-line’ monitoring with minimal reagent consumption.
Experimental
Preliminary Studies—stage I
Preliminary (stage I) studies took place using standard FIA
instrumentation (see Fig. 1(a)) in order to optimise the chemistry of
the system. A peristaltic pump (Gilson Minipuls 312, Villiers,
France) was employed to deliver the sample and reagents through
0.8 mm id poly(ether ether ketone) (PEEK) tubing, at a total flow
rate of 2.1 mL min21. The manifold included a manual sample
injection valve, model 7125, (Rheodyne, Cotati, CA, USA) fitted
with a 120 mL PEEK injection loop for the introduction of the
samples. The injected sample and carrier stream merged with the
1,10-phenanthroline solution at a T-piece connection and were
mixed in a 100 cm long mixing coil of PEEK tubing prior to
meeting with the hydrogen peroxide solution through a second Tpiece connector. The distance between the second T-piece and the
detector flow cell was kept as short as possible (approximately 50
mm) as the chemiluminescence reaction occurred almost instantaneously. The flow cell itself consisted of 65 cm of 0.6 mm id
transparent, flexible polyethylene (PE) tubing which was spiralled
to a diameter of 25.4 mm and fixed to the window of a
photomultiplier tube (PMT) (detailed below) and housed within a
light tight box. Other details have been described previously by
Coale et al.8
Micro-column
A 0.8 cm long, 0.3 cm id micro-column packed with PRP-X800
itaconic acid functionalised 20 mm PS-DVB resin (Hamilton
Company, Reno, NV, USA) was used for Cu(II) selectivity studies.
The column was incorporated in the various manifolds prior to the
introduction of the sample into the reagent streams. Back-pressure
from the 0.8 cm column was sufficiently low not to affect
performance of the peristaltic pumps used.
Reagents
In this study, all reagents were of analytical reagent grade and
contained negligible concentrations of trace metals unless stated
otherwise. Water obtained from a Milli-Q (Millipore) water
purification system was used throughout this work. All the
solutions were degassed using sonication and filtered through a
0.45 mm nylon membrane filter from Gelman Laboratories
(Michigan, USA) prior to use. Under optimal final conditions the
following reagents were prepared; Reagent 1; Deionised (Milli-Q)
water containing 0.07 mM 1,10-phenanthroline made from a 12
mM stock solution, which was found to be stable for several days
(obtained from BDH Laboratory Supplies, Poole, England and
further purified by recrystalisation with nitric acid), 0.10 mM
cetyltrimethylammonium bromide (CTAB) (BDH Laboratory
Supplies, Poole, England) and 0.075 M sodium hydroxide (Sigma
Aldrich Ltd., Dublin, Ireland). This solution was made up fresh
each day. Reagent 2; A 5% w/v hydrogen peroxide (Sigma Aldrich
Ltd., Dublin, Ireland) solution was prepared daily.
All Cu(II) standard solutions were prepared freshly each day
from stock solutions (atomic absorption spectroscopy standard
solution (Sigma Aldrich Ltd., Dublin, Ireland)) (1000 mg L21)
stored in 1% nitric acid. Calcium chloride (obtained from Sigma
Aldrich Ltd., Dublin, Ireland) and magnesium chloride (Sigma
Aldrich Ltd., Dublin, Ireland) were used to make up a pseudo
sample matrix to investigate selectivity. Other metals used for
interference studies included zinc, manganese, lead, nickel, cadmium and cobalt. All of these were atomic absorption spectroscopy
standard solutions stored in 1% nitric acid and were obtained from
Sigma Aldrich Ltd., Dublin, Ireland. Normal precautions for trace
metal analysis were taken including acid washing of all glassware
and plastic containers.
Micro-fluidic manifold fabrication
Fig. 1 Schematic diagrams and dimensions of the FIA and micro-fluidic
manifolds developed. Chip dimensions = 7.0 3 7.0 3 0.8 cm.
The micro-fluidic manifolds were designed using CAD 3D
Excalibur software. Following this, the manifold could be fabricated in one of three different ways; hot embossing from a micromilled brass master, direct micro-milling into the PMMA chip or
laser ablation. The first technique used a brass master that was
fabricated by high precision micro-milling the channels of various
dimensions. This was accomplished using a Datron 3-D M6 MicroMachining Centre, (Datron Technology Ltd., Milton Keynes, UK).
The resultant master was then used to hot emboss the PMMA chips
using a Model Hex02 Hot Embosser (Jenoptik Mikrotechnik,
Germany). In this case, the brass master was heated to 138 °C and
the PMMA was heated to 125 °C, just above the glass transition
temperature (Tg) of PMMA (105 °C). Using a controlled force
(1000 N) the master was used to emboss the PMMA. This
procedure typically took 15 min. A top plate of PMMA of equal
width was then drilled to make the reagent inlet holes and this was
bonded to the embossed plate at 130 °C and 500 N in a Class 1000
clean room.
The second technique used involved direct milling of the PMMA
substrate. In this case, the micro-milling system was used to cut the
micro-fluidic channels (200 3 200 mm) directly into the PMMA.
Although direct micro-milling removed the additional hot embossing fabrication step, the surface of the channels was not as smooth
Lab Chip, 2004, 4, 384–390
385
as that produced by the hot embossing method. The device was then
bonded to a pre-drilled top plate as previously described above.
The third method of fabrication used in this study was laser
ablation. A Excimer laser (Optec Micro-Master System, Optec
S.A., Frameries, Belgium) was used to make the micro-fluidic
channels in the PMMA (The laser mask size was set to 250 mm,
with a laser energy density of 1358 mJ cm22 and a frequency of 50
Hz, resulting in a machining speed of 0.250 mm s21). The manifold
was bonded as before.
730 mm in length with an internal width and depth of 250 mm 3 250
mm (triangular cross-section), and 210 mm in length and 250 mm 3
250 mm in width and depth for the reaction/detection channel. This
meant the theoretical approximate internal volume of the reaction/
detection channel was between 16 and 22 mL. Chemiluninescent
detection was monitored with a PMT as with the stage II instrument
described above.
Micro-fluidic manifold and instrumentation—stage II
Preliminary studies—stage I
For the initial micro-fluidic manifolds (stage II) both reagents and
sample carrier were again delivered using a standard peristaltic
pump (Gilson Minipuls 312, Villiers, France) delivering the sample
and reagents into the plastic manifold via connecting 0.8 mm inner
diameter poly(ether ether ketone) (PEEK) tubing, glued into
reagent inlet holes (1 mm diameter) on the underside of the plastic
chips, at individual flow rates of 0.06 mL min21 (0.18 mL min21
total). The sample was introduced into the carrier stream using a
manual sample injection valve, model 7125, (Rheodyne, Cotati,
CA, USA) fitted with a 3 mL PEEK injection loop. The manifold
itself had a mixing channel of 1000 mm in length, < 900 mm in
depth and 1000 mm in width, and a detection channel 190 mm in
length and again < 900 mm in depth and 1000 mm width.
Chemiluminescence was continuously measured using a PMT
(Hamamatsu Photonics HC135–01 series with R1924 bi-alkali
tube). The PMT was approximately 135 mm in height and the
window of the detector was 25.4 mm inner diameter. In this work,
the chemiluminescence occurred between 445 and 450 nm, and the
PMT spectral range was from 300 nm to 650 nm (absorbance of
PMMA is negligible above 400 nm). The PMT operated in photon
counting mode and the signal was recorded on a computer and data
processed using Microsoft Excel. The PMT was placed over the
detection channel and was secured tightly to prevent stray light
from reaching the detector. The complete micro-fluidic manifold
and PMT assembly was wrapped several times in aluminium foil to
reduce the dark count rate and enclosed in a light tight box. Fig. 1(b)
shows the schematic diagram of this initial system.
Initial studies took place using standard FIA in order to optimise the
chemistry of the system and to ascertain the analytical performance
characteristics that could be later compared to the micro-fluidic
method. Using this system three of the reaction variables were
optimised, the concentration of hydrogen peroxide, 1,10-phenanthroline and the concentration of surfactant.
Micro-fluidic manifold and instrumentation—stage III
For the final micro-fluidic system (stage III) developed, three
micro-peristaltic pumps with 0.51 mm id peristaltic tubing
(manufactured by BVT Technologies, Euro-link Associates, Tyne
& Wear, England) were used to drive the reagents and sample
streams through the micro-fluidic manifold via connecting 0.13
mm id PEEK tubing, glued into reagent inlet holes (1 mm diameter)
on the underside of the plastic chips. Each micro-pump weighed
only 34 g and was 60 mm in height and only 16 3 16 mm at the
base. These pumps were operated using a DC supply at 3 V and
were connected together using a custom made interface driven by
one PC connection. When connected in this manner it was possible
to set each flow rate independently for each pump in the range of
0.5 to 200 mL min21. Under optimal conditions for this study,
individual flows rates of 25 mL min21 (75 mL min21 total) were
used.
The final version of the micro-fluidic manifold design is
illustrated schematically as Fig. 1(c). The micro-fluidic manifold
itself was manufactured from PMMA. A number of different
designs and dimensions were investigated but the basic design
consisted of two inlets for the sample and 1,10-phenanthroline
solutions, which were subsequently mixed together in the mixing
channel. This provided the mixing necessary for the reaction
between the Cu(II) in the sample and the 1,10-phenanthroline to
take place. A third inlet at the end of the mixing channel prior to the
detection/reaction channel was used for the introduction of the
hydrogen peroxide. At the end of the detection channel there was an
outlet leading to waste. For the final laser ablated version of the
micro-fluidic manifold the dimensions of the mixing channel were
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Lab Chip, 2004, 4, 384–390
Results and discussion
Optimisation of hydrogen peroxide
Concentrations of 1,10-phenanthroline, CTAB and NaOH were
kept constant at 0.06 mM, 0.1 mM and 0.075 M respectively, while
the concentration of hydrogen peroxide was varied. From this work
it was found that a 5% hydrogen peroxide solution resulted in a
higher analytical signal for a Cu(II) standard than higher hydrogen
peroxide concentrations, with little difference in background noise,
even though in some of the literature a 10% hydrogen peroxide
solution was used.9 The actual signal to noise ratio for the 5%
hydrogen peroxide was 13.2 compared to 8.4 for the 10% peroxide
solution. The peroxide solution was prepared daily to avoid a
decrease in sensitivity that resulted from reagent instability over
time. No significant reductions in detector response when using a
single hydrogen peroxide solution for a period of 12 h.
Optimisation of 1,10-phenanthroline
Using the optimised 5% hydrogen peroxide concentration, the
concentration of 1,10-phenanthroline was then optimised. The
concentrations of CTAB and sodium hydroxide again remained
constant at 0.1 mM and 0.075 M, respectively. A 0.03 mM
1,10-phenanthroline solution was found to be optimal, producing a
significant reduction in background noise compared to higher
levels. The signal itself due to Cu(II) was also reduced but the signal
to noise ratio for the 0.03 mM 1,10-phenanthroline solution was
27.63 compared to 8.86 for a 0.06 mM 1,10-phenanthroline
solution. The 1,10-phenanthroline was also purified by recrystilisation with nitric acid as the Cu(II) signal was enhanced considerably
(by approximately 40%) when the purified 1,10-phenanathroline
was used and noise further decreased. Purified 1,10-phenanthroline
was used in all future work.
Optimisation of surfactant
Previous work by Yamada and Suzuki10 investigated the effect of
different surfactants on the Cu(II)–1,10-phenanthroline chemiluminescent reaction. It was found that an increase in signal
resulted from the addition of a cationic surfactant, while anionic
and nonionic surfactants had no significant effect. In this work,
several surfactants including Triton X-100, sodium dodecylsulfate
(SDS) and CTAB were investigated. It was found that Triton X-100
(non-ionic) and SDS (anionic) did not increase the signal and the
background noise was dramatically increased. The addition of low
concentrations ( < 0.1 mM) of the cationic surfactant CTAB was
found to dramatically reduce some of the background noise whilst
not affecting the analytical signal for Cu(II). A concentration of
0.05 mM CTAB produced a signal to noise ratio of 19.9 compared
to 6.4 for a 0.10 mM CTAB solution.
The optimal reaction pH was determined by Coale and coworkers to be between 9.8 and 10.18. In this work, the reaction pH
for Cu(II) analysis under optimised conditions was found to be
10.35. Using this system, Cu(II) could be easily determined at levels
as low as 1 mg L21 and the response was linear (n = 5, standards
injected in triplicate) over the concentration range 1 to 50 mg L21,
producing a R2 value of 0.995 (see Fig. 2(a)).
Selectivity studies
Previous work has shown the high selectivity of the Cu(II)–
1,10-phenanthroline chemiluminescent reaction, which shows little
or no response to excess concentrations ( > 10 mg L21) of alkali
metals or Cr(III) and (VI), Mn(II), Ni(II), Co(II), Cd(II), Pb(II), Al(III),
and Fe(II) and Fe(III).7,10 However, it has been reported that high
concentrations of Zn(II), Ca(II) and Mg(II) can cause minor
interference at the above concentration. To further increase the
selectivity of the developing method, a Cu(II) selective itaconic acid
functionalised resin (20 mm resin size) micro-column was investigated. Itaconic acid is a dicarboxylic acid capable of acting as a
weak cation exchanger and/or a strong chelating ion exchanger,
which within a 0.3 3 0.8 cm micro-column at pH 2 to pH 4
completely retained Cu(II) whilst showing no retention of alkali and
alkaline earth metal ions, or Mn(II), Cd(II), Co(II), Zn(II), Pb(II) and
Ni(II). By selectively removing Cu(II) from the sample matrix using
the above micro-column on-line, followed by its subsequent
elution, it was possible to remove the above potential interferences.
To illustrate this, a complete set of Cu(II) standards over the range
1 mg L21 to 1 mg L21 were made up in a sample matrix containing
10 mg L21 Ca(II) and 10 mg L21 Mg(II) (typical concentrations
found in drinking waters), whilst a second set was made up in MilliQ water only. The standards containing Ca(II) and Mg(II) were each
passed through the micro-column (column buffered to pH 4) and
the Cu(II) was selectively retained. The Cu(II) was subsequently
eluted with an equal volume of 100 mM nitric acid. Comparison of
the eluted Cu(II) standards with those prepared and analysed
directly in Milli-Q water would show how the column could be
used for complete retention and elution of Cu(II) from samples
containing high levels of Ca(II) and Mg(II).
Fig. 3(a) shows the resultant peaks for the Cu(II) standards,
which were separated from the sample matrix containing Ca(II) and
Mg(II) by the itaconic acid column. The results for Cu(II) standards
made up in Milli-Q water over the same concentration range of 1 mg
L21 to 1 mg L21 are show as Fig. 3(b). It can be seen that there was
effectively 100% separation and recovery of the copper from the
sample matrix using the itaconic acid micro-column. This means
the micro-column could also be used for preconcentration of Cu(II)
from complex sample matrices should the need arise. From Fig. 3
it is also interesting to note that Milli-Q water itself gave a small
response equivalent to < 1 mg L21 Cu(II) when analysed directly
and that this response was eliminated after treatment with the
micro-column. This would indicate that some potentially interfering species (other than Cu(II)) in the Milli-Q water itself was
causing a slight positive signal within the blank.
Initial micro-fluidic work—stage II
The first micro-fluidic manifold in this study was manufactured in
PMMA using the micro-milling and hot embossing facility. The
first manifold fabricated had a mixing channel of 1000 mm in
length, 900 mm in depth and 1000 mm in width. However, bonding
the top plate to the lower design plate causes considerable reduction
in the actual depth of the channels in the finished manifold,
particularly with wide channels such as these, and so the exact
channel depth in the bonded manifold could not be ascertained.
This early design provided the same mixing channel length as the
Fig. 3 Detector responses obtained for (a) Cu(II) standards prepared in a
matrix of 10 mg L21 Ca(II) and Mg(II) and passed through itaconic acid
mini-chelating column followed by elution with 100 mM HNO3, and (b)
Cu(II) standards prepared in Milli-Q water only.
Fig. 2 Detector responses to Cu(II) standards obtained using (a) standard FIA manifold compared to (b) initial hot embossed design (1000 3 900 mm
channels) and (c) laser ablated manifold (250 3 250 mm channels). Standard concentrations shown in mg L21.
Lab Chip, 2004, 4, 384–390
387
standard FIA method and was found to be more than adequate for
the required complete mixing of the Cu(II) in the sample and the
1,10-phenanthroline. The inlet for the introduction of the hydrogen
peroxide was placed immediately prior to the detection/reaction
channel as the chemiluminescent oxidation reaction was known to
be almost instantaneous.
A series of experiments were again carried out in order to
optimise the concentrations of 1,10-phenanthroline and CTAB
under the new flow conditions. The experimental space was defined
by varying systematically both the concentrations of 1,10-phenanthroline and CTAB from 0.01 mM to 0.10 mM. In all of the
experiments, the following conditions were used; a total combined
flow rate of 180 mL min21, sample injection volume of 3 mL, 5%
hydrogen peroxide solution and 0.075 M sodium hydroxide. From
this work the highest signal to noise ratio was achieved when using
0.07 mM 1,10-phenanthroline and 0.06 mM CTAB. Using these
new conditions, linearity and response was again investigated, and
the peaks obtained for a range of Cu(II) standards between 10 and
1000 mg L21 can be seen in Fig. 2(b). As can be seen from the figure
shown, linearity (if based upon peak height) was restricted to < 100
mg L21 Cu(II). However, as can be seen, given the much reduced
injection volume, analyte sensitivity was much improved, despite
the large reduction in the size and volume of the reaction/detection
coil. There was also very little change in background noise, giving
a detection limit of approximately 6 mg L21 (using 3 3 baseline
noise criterion). The repeat injection of a Cu(II) standard solution
using this system (n = 6) gave a % RSD value of 2.2 based upon
peak height.
Investigation of mixing process
In an attempt to improve method linearity and further miniaturise
the manifold the impact of the dimensions of the mixing channel
and reaction channels was investigated. Coloured dyes were used in
place of the reagents to investigate the mixing process, in order to
reduce the length of the mixing chamber. It was found that
complete mixing of the two streams occurred after approximately
700 mm. It was decided, therefore, that the length could be
decreased by nearly one third of the initial length (1000 mm to 700
mm). An investigation on the length of the reaction/detection
channel was also carried out in order to determine the optimal
length. Using a 100 mg L21 Cu(II) standard, the detection channel
was increasingly masked from the PMT detector to ascertain the
effect of shortening the reaction channel by 25, 50 and 75%
(equivalent to 143, 95 and 48 mm respectively). The resultant
signals for the 100 mg L21 standard can be seen in Fig. 4. During
this experiment it was found that masking the last 25% of the
reaction/detection channel, the chemiluminescent signal for the
Cu(II) standard decreased (by more than half). It was also found that
masking the last 75% of the channel led to the signal being totally
lost. This meant that although the chemiluminescence reaction was
thought to occur almost instantaneously, maximum emission took
place in the latter part of the detector channel due to a short time
delay to facilitate mixing of the hydrogen peroxide and the sample/
reagent flow. As a result, the length of the reaction channel could
not be decreased and was subsequently increased (see stage III
below).
Further miniaturisation—stage III
Taking the above results into account, the design for the final (stage
III) micro-fluidic manifold was finalised. As above, a manifold was
prepared using micro-milling to produce a brass master, followed
by hot embossing into PMMA. The final design, as shown in Fig.
1(c), utilised three individual micro-pumps to supply the reagent
stream, hydrogen peroxide stream and sample stream. To further
simplify the system, the sample injection valve was removed and
the sample fed directly into the mixing channel itself. This would
result in a continuous detector response rather than the previous
transient signal. For introduction of new sample and standard
solutions the sample pump was stopped whilst solutions were
changed. It was found that this manual procedure did not introduce
air into the system (as is evidenced by Fig. 5), although for future
on-line work a low-pressure switching valve could be readily
incorporated into the system for this purpose. The mixing channel
was reduced in length to 700 mm, while the detection channel was
increased to 210 mm. In addition, the shape of the detection channel
was optimised (fitted exactly to shape of PMT window) to
maximise sensitivity and reduce background noise. The channels
themselves were significantly narrower than the channels of the
larger chip, being 200 mm wide by 200 mm deep (although again
due to bonding the ultimate depth of the channels was significantly
< 200 mm). As in each previous case the reagent concentrations
were re-optimised to suit the new manifold design. The highest
signal to noise ratio was achieved when using 0.07 mM 1,10-phenanthroline and 0.1 mM CTAB, with other conditions set at a total
combined flow rate of 76 mL min21, 5% hydrogen peroxide
solution and 0.075 M sodium hydroxide.
Flow rates
The flow rates used in the stage III micro-fluidic system were each
controlled by a separate micro-pump, however all the reagents were
pumped at the same flow rate. Briefly, the effect of flow rate on
response was investigated using individual flow rates of 0.029,
0.025 and 0.021 mL min21 for each of the three streams (measuring
in total 0.086, 0.076 and 0.064 mL min21). Results showed that the
analytical signals were slightly increased by decreasing flow rates
(see Fig. 5(a)). However, although the lowest flow rates (0.021 mL
min21 for each stream) produced the highest absolute signal, the
background was also noisier, and therefore individual flow rates of
0.025 mL min21 was found to be optimum.
Linearity
Using the optimised conditions, a series of measurements was
performed with Cu(II) standard solutions ranging from 0 to 50 mg
L21. A stepwise graph was produced over this concentration range
and is shown as Fig. 5(b). The resulting signal heights were plotted
as a function of concentration and the results were found to produce
an excellent linear correlation within this given concentration range
(R2 = 0.993, n = 6, standards measured in duplicate). Using the
micro-fluidic manifold, Cu(II) could be easily determined at
concentrations as low as 10 mg L21, with the S/N ratio of 3.
Fig. 4 Detector responses obtained for a 100 mg L21 Cu(II) standard with
increasingly reduced reaction/detection channel length (equivalent to 190,
143, 95 and 48 mm).
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Lab Chip, 2004, 4, 384–390
Comparison of fabrication techniques
Three alternative micro-fabrication techniques were investigated,
namely hot embossing into PMMA, direct micro-milling of the
channels into the PMMA itself and thirdly the use of laser ablation.
Each of these techniques result in different channel profiles and
surface morphologies. Laser ablation results in a characteristic
shallow ‘V’ shaped channel and direct micro-milling into the
PMMA results in a rougher surface than the hot embossed
manifold. For direct comparison with the hot embossed manifold,
detector response and linearity was determined on each manifold
using identical reagent concentrations and flow rates. The directly
micro-milled manifold resulted in the largest comparative unit
response, with a calibration slope equal to ~ 751 counts s21 for
each mg L21 of Cu(II) compared to ~ 378 counts s21 for each mg
L21 Cu(II) for the above hot embossed manifold. However,
background noise was also increased. The results using the directly
micro-milled manifold were found to be linear up to 100 mg L21 (R2
= 0.983, n = 5, standards injected in duplicate).
The laser ablated manifold was found to give the lowest unit
response with the lowest calibration slope, only 61 compared to the
above values. However the manifold also produced the most linear
response over the greatest concentration range 0–150 mg L21 (R2 =
0.998, n = 5, standards injected in duplicate, see Fig. 2(c)). In
addition, the laser ablated manifold also gave the lowest background noise, resulting in a detection limit of approximately 20 mg
L21 Cu(II). The reason for the lower response and noise with this
manifold is simply related to the fact that the laser ablation process
results in the shallow ‘V’ shaped channels which effectively reduce
the channel volumes considerably ( ~ 50%), compared to the
alternative fabrication methods. To partially compensate for this
profile the laser ablated channels were cut 250 mm wide by 250 mm
deep, hence the reaction/detection channels of the ablated manifolds was calculated to be within the range 16–22 mL compared to
21–30 mL for the hot embossed manifold. Fig. 6(a) shows the
comparative responses for each of the above micro-fluidic
manifolds for increasing Cu(II) standard solutions (Milli-Q water
was introduced between each standard reading). Fig. 6(b) shows the
reproducibility of the laser ablated manifold with the repeated
analysis (n = 5) of a 75 mg L21 Cu(II) standard.
Sample carry over
Fig. 6 shows recovery time (time taken for a return to baseline
signal following removal of the sample) for each of the microfluidic manifolds was in the order of 2–3 min. This indicates some
degree of adsorbance of the reagents onto the channel walls, which
may be slowly washed off during the Milli-Q washing step.
However, as Figs. 5(b) and 6(a) show, the system does return to the
starting baseline when allowed sufficient recovery time, and
although this may increase individual sample analysis time, for
Fig. 5 Detector responses obtained using the hot embossed micro-fluidic manifold (200 3 200 mm channels). (a) Effect of flow rate upon response, (b)
system linearity, and (c) on-line analysis of drinking water. All concentrations shown in mg L21.
Fig. 6 (a) Comparison of detector response for three micro-fluidic manifolds, (1) laser ablated manifold (250 3 250 mm), (2) hot embossed manifold (200
3 200 mm), (3) direct micro-milled manifold (200 3 200 mm). (b) Repeat analysis of 75 mg L21 Cu(II) standard using laser ablated manifold. (c) Analysis
of SRM 1640 standard reference freshwater sample. Milli-Q water blanks run between each sample/standard analysis.
Lab Chip, 2004, 4, 384–390
389
Table 1 Analytical performance data for developed methods
Fabrication method
Flow rates/
mL min21
reagent
Linear
rangea/
mg L21
Slope
Standard FIA
Hot embossing 1000 3 900 mm
Hot embossing 200 3 200 mm
Micro-milling 200 3 200 mm
Laser ablation 250 3 250 mm
0.7
0.06
0.025
0.025
0.025
1–50
< 100
20–100
40–100
25–150
726
—
751
378
61
a
Approx.
detection
limit/mg L21
0.995
—
0.996
0.983
0.998
1–2% (n = 3)
2.2% (n = 6)
2–3% (n = 3)
4–5% (n = 2)
2–3.5% (n = 5)
1
6
10
10
20
Data based upon peak height measurements.
longer term on-line monitoring purposes this should not be a
significant problem.
Table 1 shows the comparative analytical performance data
determined for the three micro-fluidic manifolds, and those data
obtained for standard FIA and the initial larger scale hot embossed
manifold. The data shown were obtained under the specific reagent
conditions used within this study only.
Analysis of real samples on-line drinking water analysis
Using the hot embossed micro-fluidic manifold and conditions
described above, the on-line analysis of drinking water was
undertaken. The drinking water from a laboratory tap was
continuously fed via an in-line filter to the sample inlet of the
manifold. Within the sample line a switching valve was placed
which allowed the tap water to either by-pass or be passed through
the Cu(II) selective itaconic acid micro-column (pre-buffered to pH
4) detailed earlier, allowing verification that the detector response
was due to Cu(II) only. The results of this experiment can be seen
in Fig. 5(c). As can be seen, the concentration of Cu(II) found within
the laboratory tap supply was approximately 80 mg L21. On-line
passage of the tap water through the itaconic acid column
completely removed the chemiluminescent signal, indicating that
the sample matrix was not causing significant interference in this
application.
Analysis of certified reference water sample
To check for method accuracy a standard reference material (SRM)
from the National Institute of Standards and Technology (NIST)
was analysed for Cu(II). The sample (SRM 1640) was composed of
natural fresh water (river) had been filtered and acidified with 0.5
M nitric acid. This sample was certified to contain 85.2 mg L21 ±
1.2 mg L21 Cu(II). The sample was neutralised using sodium
hydroxide and was diluted by 50% with Milli-Q water. This sample
was then analysed using the laser ablated manifold. Firstly, 50 and
100 mg L21 Cu(II) standards were analysed, with a Milli-Q water
blank run between standards. The 50% dilution of the NIST
standard was then analysed. The resultant signals can be seen in
Fig. 6(c). This sample was found to contain approximately 40 mg
L21 Cu(II) which corresponds to ~ 80 mg L21 in the SRM, within
390
R2 value
Reproducibility
% RSD
(no. replicates)
Lab Chip, 2004, 4, 384–390
±6% of the true value, representing excellent accuracy for a microfluidic device when analysing a complex freshwater sample for
trace Cu(II).
Conclusions
The development of a micro-fluidic manifold and analytical
method for Cu(II) determinations has been described. The results
have shown that the standard FIA method can be effectively
reduced to the micro-fluidic format whilst maintaining acceptable
linearity, precision and accuracy and with appropriate detection
methods such as chemiluminescence, also maintaining excellent
sensitivity. The micro-fluidic system developed can be readily
made portable or be used for on-line monitoring with as little as 3.0
mL h21 total reagent consumption.
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