High-sensitivity online detection for microfluidics via cavity ringdownspectroscopy

Dean James,a Bobby Oag,a Cathy M. Rushworth,a Jason W. L. Lee,a Joanna Davies,b Joao T. Cabralb and

Claire Vallance*a

Received 23rd February 2012, Accepted 2nd April 2012

DOI: 10.1039/c2ra20349a

We report the coupling of cavity ringdown spectroscopy (CRDS) with a microfluidic chip fabricated

using a rapid prototyping method, in order to demonstrate high-sensitivity, non-contact online

detection in microfluidics. Conventional UV-vis absorption techniques are largely ineffective for

microfluidic detection due to the small sample volumes and short path lengths. The multipass

absorption achieved in cavity ringdown spectroscopy increases the effective absorption pathlength by

several orders of magnitude, and hence enhances the detection sensitivity. A cavity ringdown

spectrometer, operating at a single wavelength of 532 nm for the purposes of the proof-of-concept

measurements presented here, has been developed for online detection on a polymer/glass microchip

fabricated by frontal photopolymerisation. High sensitivity absorption measurements on liquid

samples with volumes of tens to hundreds of nanolitres and absorption pathlengths ranging from tens

to hundreds of microns are demonstrated. A series of proof-of-concept experiments show that the

technique has the ability to monitor both static and time-varying analyte concentrations. Firstly, the

detection limit of the system is estimated from a three-standard-deviation error analysis of absorption

measurements made on dilute aqueous solutions of potassium permanganate (natural absorption

coefficient (4805 ¡ 10) M21 cm21 at 532 nm). The detection limit was found to be y210 nM for a

466 mm pathlength, corresponding to an absorption of 1.0 6 1023 cm21. Online pH measurements on

a 20 nL sample are performed by monitoring the absorption of phenolphthalein indicator present at

millimolar concentrations. Finally, CRDS has been applied, for the first time, to monitoring chemical

reaction kinetics on a microfluidic chip, tracking the oscillation period of the well-known Belousov–

Zhabotinsky reaction.

1 Introduction

Detection and quantification of the tiny volumes of chemical

species produced in microfluidic systems remains a major

challenge to the full exploitation of microfluidics in biochemical

synthesis and analysis. Numerous authors have identified the

issue of ‘‘efficient extraction and utilisation of the vast amounts

of information produced’’ in microfluidic experiments.1–3

Droplet-based microfluidics have brought a paradigm shift in

high-throughput experimentation. Each droplet represents an

independent ‘‘reactor’’ volume,4,5 and the ability to produce

droplets at up to kHz rates opens up a vast number of

applications in biological, chemical and materials synthesis.

However, such approaches place additional demands on the

detection methods employed, as any detection scheme must be

commensurate with these high droplet production rates. At

present, chemical analysis is often carried out off-chip6 using

conventional laboratory techniques, thus becoming the rate

limiting step in what would otherwise be a high-throughput

microfluidic reactor.

Optical detection provides an appealing approach for online

measurements of microfluidic systems. Several optical and

spectroscopic on-chip detection methods have thus been

integrated with microreactors in recent years to permit the

spatio-temporal mapping of reactions and their optimisation,

and are the subject of several recent reviews.3,7–10 Laser-induced

fluorescence provides a powerful approach for rapid, in situ,

detection due to its high sensitivity and small mass requirements,

which are well suited to droplet analysis.4,5 Advances in

fluorescence lifetime imaging (FLIM) have permitted flow

mapping within droplets with 1 ms temporal resolution.11,12

However, such approaches are restricted in scope since they

require the presence of fluorophores, and the labelling of

reagents is not always possible or is undesirable; for example,

in reaction screening and discovery.

UV-vis spectroscopy;13–15 Fourier transform infrared spectro-

sopy (FT-IR),13,16–18 including multiple-internal reflection

aDepartment of Chemistry, University of Oxford, Chemistry ResearchLaboratory, 12 Mansfield Rd, Oxford, OX1 3TA, UK.E-mail: claire.vallance@chem.ox.ac.ukbDepartment of Chemical Engineering, ACE 311A, Imperial CollegeLondon, London, SW7 2AZ, UK

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(MIR)16 and imaging;18 Raman scattering;13,20–22 microcoil

nuclear magnetic resonance (NMR)19,23,24 or stripline detec-

tion25 with microcoils; and surface plasmon resonance (SPR),

have all been reported as approaches to online detection in

microfluidics. In general, these approaches involve a high degree

of spatial averaging over large detection areas (for example, in

plane optical detection26) or integration over relatively long

times17,22 in order to acquire statistically significant spectro-

scopic data. They are therefore suitable for single phase,

continuous reactions with stationary (time-invariant) composi-

tion profiles, but are not generally applicable to multiphase

flows. Recently, surface-enhanced Resonance Raman spectro-

scopy (SERRS) has been reported to achieve sub-millisecond

time resolution with high sensitivity on droplets containing

magnetic nanoparticles,27 and progress in FT-IR imaging

has enabled the label-free resolution of segmented flows with

y100 ms time resolution.18

Advanced UV-vis absorption techniques are particularly

attractive for microfluidic detection due to their universal nature

and potential for high sensitivity: every analyte absorbs light in

some region of the electromagnetic spectrum, and by selecting a

wavelength range across which the solvent is transparent and

there are no significant overlapping absorption bands from other

species, a sufficiently sensitive absorption measurement can

allow for label-free analyte detection. However, minute detection

volumes (pL–nL) and short path lengths (typically a few hundred

microns or less) severely limit the application of traditional

single-pass absorption spectroscopy in microdevices. Cavity

ringdown spectroscopy (CRDS) is a highly sensitive absorption

technique based on the decay of light within a high finesse

optical cavity.28 Most CRDS measurements, including ours,

employ a simple confocal Fabry–Perot cavity, comprised of two

highly reflective concave mirrors (reflectivity . 99.8%). A pulse

of laser light is directed into the back face of the first mirror and

a small amount couples through the mirror into the cavity. The

light is repeatedly reflected back and forth between the mirrors,

decaying exponentially with time as a constant fraction couples

out from the cavity on each interaction with the mirrors. The

decay time constant, or ‘ringdown time’, t is determined purely

by the geometry of the cavity and the round-trip transmission T:

t0~ncd

c({ ln T)(1)

where d is the cavity length (defined as the centre-to-centre

distance between the front faces of the two cavity mirrors), nc is

the refractive index within the cavity medium, and c is the speed of

light. For a two-mirror cavity, the cavity loss L = 1 2 T is

determined simply by the reflectivity of the mirrors. However,

inserting a microfluidic chip into the cavity introduces increased

scattering and reflection losses at the surface boundaries, which

must also be included in L. When an absorbing sample is

introduced into the cavity, absorption by the sample increases the

cavity losses, reducing the ringdown time, which is now given by:

t~ncd

c0({ ln TzaCl)(2)

where a is the absorption coefficient, C the concentration, and l is

the single-pass path length through the sample. A comparison

between the ringdown times recorded in the absence and presence

of a sample yields a quantitative determination of the sample

absorption.29 The absorption per unit pathlength, k = aC can be

shown to be:

k~aC~ncd

cl(1

t{

1

t0) (3)

CRDS and related techniques are currently amongst the most

sensitive spectroscopic absorption techniques available for both

the gas-phase30 and the liquid-phase,31,32 although they have

been much more widely applied in the gas-phase. The high

sensitivity results firstly from the vast increase in optical path

length relative to single pass techniques, and secondly from the

fact that because the technique relies on a measurement of the

rate of decay of light intensity within the cavity rather than on

the intensity itself, the signal is decoupled from the initial light

intensity and is therefore largely immune to noise arising from

shot-to-shot variations in the light source. While the method is

relatively insensitive to fluctuations in the incident light intensity,

the source intensity is one of the key factors that determine the

signal-to-noise ratio of the ringdown signal, and therefore the

accuracy to which the ringdown time can be determined.

The high sensitivity of CRDS makes it an attractive option for

microfluidics applications, providing a vastly increased optical

pathlength without increasing the sample volume probed. To our

knowledge, most attempts to couple CRDS with microfluidic

systems have so far employed optical fibre loop cavities rather

than Fabry–Perot cavities.33 This approach has the advantage

that optical fibres are well matched in size to the dimensions of

microfluidic channels, with fibre core diameters commonly less

than 500 mm. However, fibre-loop CRDS has relatively high

intrinsic cavity losses,34 resulting in a reduced sensitivity. Here

we describe the application of conventional two-mirror CRDS to

the interrogation of microfluidic liquid samples. CRDS measure-

ments on liquid samples are inherently less sensitive than gas-

phase measurements due to increased scattering and absorption

losses associated with both the sample and its container, but still

represent a considerable improvement on single-pass methods.

While liquid can be introduced to fill the entire space between the

cavity mirrors,35–38 the requirement of probing small liquid

sample volumes in microfluidics usually necessitates the intro-

duction of (typically glass) containers (otherwise known as

absorption cells) into the cavity. Past work on liquid-phase

CRDS has focused on the introduction of cuvettes39,40 or flow

cells41,42 into the cavity, with a particular emphasis on the use of

CRDS for small volume HPLC. The path lengths and detection

sensitivities achieved using these techniques are summarised in

Table 1 of ref. 43. The majority of liquid-phase CRDS have

probed sample volumes . 1 mL, with notable exceptions being

the work of Snyder and coworkers,41 Bechtel and coworkers42

and Alexander.43 The reader is also directed to a recent review of

liquid-phase CRDS techniques.44

We report what we believe to be the first example of CRDS

measurements on a microfluidic chip inserted into a two-mirror

cavity. The chip can be readily fabricated via rapid prototyping

and the non-contact nature of the two-mirror approach should

allow for the spatio-temporal mapping of the chip, avoiding

expensive integrated and less flexible spectroscopic probe

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arrangements. We have characterised the sensitivity of our

microfluidic-CRDS system and also include proof-of-concept

demonstrations of pH measurements and real-time tracking of

an oscillating reaction.

2 Experimental

2.1 Instrumentation

A schematic of the experimental setup used is shown in Fig. 1(a).

The light source is a pulsed Nd/YAG laser: either a Teem

Photonics NP-10620-100 (6 mJ per pulse, 900 ps pulse width,

7.4 kHz repetition rate), which was used for the detection limit

measurements, or a Teem Photonics SNP-08E-100 (8 mJ per

pulse, 900 ps pulse width, 7.4 kHz repetition rate), which was

used for the remaining experiments. In each case, the laser

output is frequency doubled to produce a beam of 532 nm light.

The optical cavity is formed from two concave dielectric mirrors

(CVI Melles Griot, reflectivity . 99.8% at 532 nm) with a

diameter of 25 mm and a radius of curvature of 1 m. For the

majority of experiments presented here, the mirror separation

was 875 mm, except for the detection sensitivity measurements

described in Section 2.2.1, for which the separation was 585 mm.

Light is coupled into the cavity by directing the laser pulse into

the rear of one of these mirrors. An iris is placed in front of the

cavity such that the beam radius in the centre of the cavity is

estimated to be around 300 mm. Light emerging from the cavity

is detected by a photomultiplier tube (PMT, Hamamatsu,

H6780-20), which is either placed directly behind the second

mirror (in which case the PMT is shrouded in blackout material

to reduce background signal caused by ambient light), or

coupled to the cavity output via a 3 mm diameter liquid light-

guide (Edmund Optics, NT53-428). The signal from the PMT is

displayed on a digital oscilloscope (Tektronix, TDS 3044B),

which is interfaced to a personal computer via a GPIB-USB

interface (National Instruments, 778927-01). In all the experi-

ments presented here, the laser pulse duration of 900 ps is less

than the round trip time of the cavity, such that the cavity output

consists of a train of pulses with exponentially decaying

intensities.

Data is acquired and analysed in real time using a home-written

LabVIEW program. The data acquisition time, corresponding to

a full ringdown trace, is typically 1 ms, commensurate with online

droplet analysis. However, the data transfer between the

oscilloscope and the computer is currently limited to four traces

per second. In a typical measurement, the ringdown trace is

averaged 512 times on the oscilloscope, and 50 averaged traces are

recorded and saved. For each trace, signal intensity and time

thresholds are applied if required to limit the data analysis to the

exponentially decaying region of the ringdown pulse train. A

peak-finding algorithm is employed to determine the baseline-

subtracted amplitude S of each peak in the pulse train, and the

ringdown time is determined from a linear fit to a log plot of the

resulting intensity vs. time data, i.e.

ln S~ ln S0{t

t(4)

where S0 is the intensity of the first fitted peak. The fitting

procedure generally returns R2 values of 0.998 or better.

The microfluidic chip is mounted in the centre of the cavity on

a double-rotation (Thorlabs, RP01) and three-axis translation

stage (Newport Corporation, 443), with micrometer resolution

actuators (Newport Corporation, SM-25), allowing individual

microfluidic channels to be precisely located within the laser

beam path. Two different designs of microfluidic chip were

employed in this work, both fabricated by rapid prototyping of a

multifunctional thiol-ene negative resist via frontal photopoly-

merisation,45–48 and yielding organic solvent resistant polymer

matrices sandwiched between two glass faces. The first liquid cell

was manufactured by sealing a 100 mm thick glass cover slip

against a 1 mm glass microscope slide which had been drilled

with inlet and outlet ports. A thiolene-based negative photoresist

(Norland Optical Adhesive, NOA 81) was applied around the

edges of the cover slip, sealing the system and resulting in a single

5 mm wide, 61 mm deep channel; this cell was employed to

make pH measurements, sampling a volume of around 20 nL

(assuming in this case a beam radius of around 320 mm). The

second type of microdevice was fabricated by sandwiching the

thiolene resist between two 141 mm thick glass cover slips,

separated by silicon wafer spacers at a distance of 380 mm. A

photomask printed on an acetate film with a negative of the chip

design (shown in Fig. 1(b)) was then secured over the top layer,

and the system was cured via frontal photopolymerisation using

a collimated UV source. A calibrated UV light dose was

delivered to ensure that the thiolene in the unmasked regions

was completely cured. The uncured photoresist was then pushed

out of the resulting microfluidic channels (at a temperature of

65 uC, to reduce viscosity) using pressurised air, followed by

acetone. The channels were inspected using an inverted micro-

scope (Olympus, IX71) to ensure that no defects or contaminants

were present. Nanoports (Upchurch Scientific) were fitted to

holes pre-drilled through one of the glass cover slips, forming

inlets/outlets capable of connection to a syringe pump. The edge

of the completed microfluidic chip was then mounted on a 1 mm

thick glass microscope slide, such that the microscope slide did

not infringe on the path of the laser, to provide a rigid point at

which the chip could be clamped securely in place inside the

ringdown cavity. Once mounted within the cavity, one or more

syringe pumps (Chemyx Inc, Fusion 400) were used to inject the

samples into the microdevice.

Incorporating a microfluidic chip into the cavity introduces

four additional surfaces at which scattering and reflection can

Fig. 1 (a) Schematic of the experimental setup (see text for details); (b)

design of the microfluidic chip used for the majority of the work

described in this paper. Inlet/outlet ports are labelled A, B, and C.

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occur, as illustrated in Fig. 2(a). In order to minimise scattering

losses, great care was taken to ensure high quality and cleanliness

of all surfaces, including the internal surfaces of the chip.

Reflection losses may be reduced either by ensuring that all

surfaces are normal to the laser beam, which in practice is

extremely difficult to achieve using our current chip fabrication

procedure, or by mounting the chip within the cavity at

approximately Brewster’s angle to the (p-polarised) laser beam.

In our setup, the chip is mounted on a rotation stage, allowing

the angle of the chip to the cavity axis to be adjusted so as to

optimise the ringdown time. Ringdown measurements on an

empty cavity indicate that the intrinsic cavity losses resulting

from transmission through the mirrors are around 0.2%. An

additional loss of typically around 0.5% is incurred by placing a

water-filled thiolene-glass microfluidic chip within the cavity at

an optimised angle of 59u (see discussion of optimisation

procedure below), reducing the measured ringdown time from

952 ns to around 272 ns.

Prior to carrying out any spectroscopic measurements, the

dependence of the ringdown signal on the chip angle within the

cavity was characterised. Fig. 2(b) shows the experimentally

measured ringdown time as a function of the angle between the

chip surface normal and the cavity axis. There is a clear

maximum in ringdown at an angle of 59u, and this chip angle was

used for all further ringdown measurements. We note that at the

optimum angle of 59u the optical pathlength through the sample

is increased significantly from the optical pathlength at normal

incidence.

The dependence of the ringdown time on chip angle may be

explained simply by considering the reflection losses at each

surface within the cavity. Fig. 2(a) shows the various interfaces

encountered by the light beam as it passes through the chip. The

angles of refraction at each interface are defined by Snell’s law:

n1sinh1 = n2sinh2 (5)

where n1 and n2 are the refractive indices of the media on either

side of a boundary, and h1 and h2 are the angles of the light beam

to the surface normals. The reflection loss at each interface can

then be modelled using the Fresnel equations:

Rp~

n1

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

1{(n1

n2sinh1)2

r

{n2cosh1

n1

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

1{(n1

n2

sinh1)2

r

zn2cosh1

2

6

6

6

4

3

7

7

7

5

2

(6)

where Rp is the proportion of p-polarised laser light reflected. To

a good approximation, the total cavity loss per pass is the sum of

the reflection losses from the chip surfaces and at the surfaces of

the two cavity mirrors. Once the total loss per pass has been

calculated in this way as a function of chip angle, eqn (1) may be

used to convert the angle-dependent losses into angle-dependent

ringdown times. The result is shown together with the experi-

mental data in Fig. 2(a). The measured and modelled curves are

in good agreement, though the optimum angle of 59u measured

experimentally is slightly higher than the 56u predicted by the

model. The discrepancy is most likely due to slight imperfections

in the chip, for example surfaces that are not precisely parallel to

each other.

2.2 Measurements

2.2.1 Detection limits. The minimum detection limit, kmin, may

be determined from the minimum detectable change Dtmin in the

baseline ringdown time, t0. Assuming tt0 # t02, we have49

kmin~ncd

cl

Dtmin

t20

(7)

In this work, we define Dtmin as three times the standard

deviation (s) in the baseline ringdown time t0. It can be seen

from eqn (7) that improving the sensitivity of a ringdown

measurement relies primarily on minimising the baseline losses to

maximise t0, as well as on increasing the pathlength of light

through the sample (l). Short pathlengths are inherent in

microfluidic systems, and significant cavity losses are unavoid-

able when including a microfluidic chip. While this will limit the

sensitivity of the system, considerable increases in sensitivity over

single pass measurements are still gained by cavity enhancement.

For comparison with the above estimate of our detection

sensitivity, the detection limits of the system were also evaluated

via absorption measurements on a series of low-concentration

aqueous solutions of potassium permanganate, KMnO4, a strongly

absorbing species whose visible absorption maximum lies close to

the wavelength of our laser (532 nm). Concentrations spanning the

range from 1024 to 1027 M were prepared by serial dilution, flowed

through the microfluidic chip mounted within the cavity at a

constant flow rate of 0.2 mL min21, and probed using CRDS as

described above. Between each measurement, the microfluidic chip

was flushed with water flowing at 0.2 mL min21 for five minutes,

and a measurement of the baseline ringdown time (t0) was obtained

Fig. 2 (a) Interfaces experienced by the light beam as it passes through

the microfluidic chip; (b) measured dependence of the ringdown time on

the incidence angle of the laser to the microfluidic chip within the cavity.

The measurements are compared with the results of the simple model

described in the text, which is based on quantifying the reflection losses at

each interface.

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to ensure that the chip was not contaminated with residual KMnO4

solution. The (natural) absorption coefficient, a, for KMnO4 at

532 nm was determined in a conventional single pass absorption

measurement (repeated five times) through a 1 cm cuvette filled

with 6.5 6 1024 M KMnO4 to be (4805 ¡ 10) M21 cm21 (Varian

Cary-100 Bio UV-vis spectrometer). Given that the 380 mm

pathlength across the chip was found to vary by several tens of

microns (measured using a micrometer), as the two chip faces were

not perfectly parallel to each other, rather than using a fit of the

ringdown data to eqn (3) to determine the a value, instead the

pathlength across the chip at the intersection point of the laser beam

was determined using a which was precisely known from the UV-vis

spectrometer data.

2.2.2 pH measurements. As a proof-of-concept, the cavity

ringdown spectrometer was used to measure the pH of a series of

buffer solutions (pH 7.6 to pH 11) through detection of the

change in absorption coefficient of the phenolphthalein indicator

at 532 nm. Phenolphthalein, shown in Fig. 3 (compound A), is

colourless below pH 8.2, but deprotonates between pH 8.2 and

10.0 to form the pink compound B, which absorbs at 532 nm.

Above pH 11, the pink colour begins to fade as compound C is

produced.

The process involved in the colour change may be summarised

as HInd > H+ + Ind2, where HInd and Ind2 are the protonated

and deprotonated forms of the indicator, respectively, with

pKa~{ log½Hz�½Ind{�½HInd� (8)

The concentration of deprotonated phenolphthalein, [Ind2],

may be determined from the results of an absorption measure-

ment, using eqn (3). Rearranging eqn (8) then allows the pH to

be determined, assuming the initial concentration of indicator is

known.

pH~pKaz log½Ind{�

½HInd�0{½Ind{� (9)

For these measurements, a stock solution of 0.015 M

phenolphthalein solution was prepared in a 1 : 1 mixture of

ethanol and water. For each measurement, 0.1 mL of indicator

was added to 5 mL of buffer solution, to give a total indicator

concentration of 2.9 6 1024 M, and the absorption of the

resulting solution was determined following a measurement of

the ringdown time t. Two types of buffer solution were used: a

sodium tetraborate and hydrochloric acid buffer solution was

used for the range of pH 7.6 to 9.4; and a glycine and sodium

hydroxide solution was used for the range from pH 9.0 to 11.0.

The absorption coefficient of phenolphthalein was determined

for each pH used.

As a reference for comparison, the experiment was repeated in a

1 cm cuvette using a commercial UV/vis spectrometer (Unicam,

UV2-100). A concentration of 1.49 6 1023 M phenolphthalein

was used for these measurements, an order of magnitude larger

than that used in the microfluidic experiment. To account for this

difference, the single-pass data was scaled by the ratio of indicator

concentrations.

2.2.3 Oscillations of the Belousov–Zhabotinsky reaction. To

demonstrate that time-resolved measurements are also possible

with our system, the Belousov–Zhabotinsky (BZ) reaction was

performed on a microfluidic chip inside the cavity. The BZ

reaction is an example of an oscillating reaction,50 in which

Ce(IV)/Ce(III) is used to catalyse the oxidation and bromination

of malonic acid by BrO32 in H2SO4. The reaction occurs in a

periodic cycle, with a clear red to blue colour change in the

presence of Ferroin indicator. Under our experimental condi-

tions, the oscillation time was several tens of seconds. When red,

the solution absorbs at our operating wavelength of 532 nm,

which allows the progress of the reaction to be monitored.

For simplicity, the reagents (5 mL of 0.23 M NaBrO3 aqueous

solution, 5 mL of a 0.31 M malonic acid and 0.059 M NaBr

aqueous solution, 5 mL of a 0.019 M Ce(NH4)2(NO3)6 and

2.7 M H2SO4 solution, with 0.1 mL of 0.0125M Ferroin

indicator)51 were mixed in bulk prior to being injected into the

chip. The reaction mixture was injected into the chip at a flow

rate of 0.1 mL min21, but once the continuous liquid flow had

passed the laser beam position, flow was stopped and the

oscillations were recorded under static conditions. Ringdown

traces were averaged 32 times on the oscilloscope, and data was

acquired at a rate of 1 Hz. The oscillations were then followed by

monitoring the change in t over time.

To obtain a reference to compare with, the experiment was

repeated at the same concentration in a 1 cm cuvette and

monitored as a function of time, via a single-pass 532 nm

absorption measurement using the same laser and a silicon

photodiode detector (Thorlabs DET 10A).

2.2.4 Droplet flow measurements. We next consider the

applicability of this approach to droplet microfluidics, with

high-throughput applications in mind.52 As mentioned above,

droplet-based microfluidics present additional challenges, parti-

cularly in the requirement of fast data acquisition rates to resolve

individual travelling droplets. In addition, we expect higher

losses than found in continuous flow because of the presence of

possible lubrication layers between microchannel, carrier fluid

and suspended phases. As a second demonstration of time-

resolved measurements using our experimental system, the two-

mirror cavity was used to monitor droplets of 1 6 1026 M

aqueous rhodamine 6G (R6G) solution. The aqueous sample

was injected into Input B of the thiolene-glass chip (illustrated in

Fig. 1(b)), and a carrier phase of toluene was injected into Input

A, resulting in aqueous plugs compartmentalised by the toluene

carrier phase. Ringdown measurements were performed in which

the flow rates were varied between 0.003 mL min21 and

0.05 mL min21 in order to vary the size of the plugs over theFig. 3 Structural forms of phenolphthalein.

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range from 2 to 10 mm and their travel velocity in the channel

over the range from 130 to 2200 mm s21.

3 Results and discussion

3.1 Detection limits

As described in Section 2.2.1, ringdown times were recorded for

a series of aqueous solutions of KMnO4, with concentrations

ranging from 1024 to 1027 M. Typical ringdown signals are

shown in Fig. 4. When used in combination with the known

absorption coefficient a for KMnO4 of (4806 ¡ 106) M21 cm21

(determined in a separate single-pass measurement using a

solution housed in a commercial cuvette) these measurements

allow us both to determine an accurate value for the pathlength

within the microfluidic chip, and to determine the limit of

detection for KMnO4. The pathlength is determined from the

gradient of a plot of k against concentration (eqn (3)), shown in

Fig. 5, to be 466 ¡ 10 mm.

The measured ringdown time for a chip filled with pure water

was 271 ns, with a standard deviation of 0.6 ns. Determining the

detection limit as described in Section 2.2.1, we have Dt = 3s =

1.8 ns, which when substituted into eqn (7) yields an absorbance

detection limit of kmin = 1.0 6 1023 cm21. This corresponds to a

concentration detection limit for KMnO4 of approximately 214 nM

in an illuminated volume of 132 ¡ 3 nL, which is in qualitative

agreement with the concentration at which a change in ringdown

time can be discerned relative to the reference measurement.

The detection limit can also be estimated from the 3s

uncertainty in the intercept of the plot shown in Fig. 5. The

value of 5.2 6 1023 cm21 obtained in this way is somewhat

higher than that determined above from the baseline noise in our

t0 measurement. This is unsurprising, as the measurement based

on t0 assumes that there are no sources of error other than the

intrinsic noise in the ringdown measurement. In reality, factors

such as a drift in the cavity alignment and uncertainties in the

analyte concentration are likely to contribute to the overall

measurement uncertainty.

While we believe our work marks one of the first applications

of CRDS to measurements on microfluidic samples, measure-

ments through a similar path length of liquid have been carried

out previously. Snyder and coworkers41 used a bespoke high-

optical-quality Brewster’s angle flow cell with a volume of 10 mL

and an optical path length of 300 mm to achieve a detection limit

of 6.2 6 1024 cm21 at 470 nm. Using the same flow cell, Bechtel

and coworkers42 further improved the detection limit to 7.8 61026 cm21 by replacing the pulsed 470 nm laser source used to

excite the cavity with a single-mode continuous wave laser source

operating at 488 nm, thus reducing noise on the signal associated

with shot-to-shot fluctuations in the laser intensity. Our

detection limit is somewhat higher than those reported by

Snyder and Bechtel, which can be ascribed primarily to the fact

that our simple microfluidic chip is not manufactured from high

optical quality components, and almost certainly suffers from

small scattering losses at the surfaces. There is scope to improve

the optical quality of the microfluidic chip, and therefore to

lower our detection limit, but even so, the integrated microfluidic

chip CRDS system provides a good general platform for

chemical analysis, with the potential for probing smaller sample

volumes than the flow cell arrangement.

The detection limit obtained here within a two-mirror cavity

can also be compared to that obtained using fibre-loop CRDS

techniques. Rushworth et al.34 recently reported a detection limit

of 0.11 cm21 using a cavity comprising a 3.08 m loop of 365 mm

core diameter optical fibre. The path length through the sample

was 180 mm, corresponding to a total probed sample volume of

19 nL. Using a similar technique,53 Waechter et al. recently

reported a detection limit of 4.6 6 1022 cm21 in a slightly

smaller volume (100 nL compared to 132 nL), but with a

significantly longer path length (800 mm compared to 466 mm). In

this case, the fibre-loop cavity comprised a 9.25 m loop of

400 mm core diameter optical fibre. While fibre-loop cavities

have a number of advantages for measurements on microfluidic

samples, being well size-matched to microfluidic channels and

inherently able to support a broad range of wavelengths, the

detection limits achievable are currently several orders of

magnitude poorer than for two-mirror arrangements due to the

unavoidably higher cavity losses associated with coupling light

into and out of the loop and introducing a sample region.

3.2 pH measurements

As explained in Section 2.2.2, CRDS measurements of the

optical absorption by phenolphthalein indicator were used to

Fig. 4 Typical ringdown traces recorded for a cavity containing a

thiolene glass microfluidic chip: reference ringdown recorded when the

channels are filled with water (grey trace); ringdown recorded when the

channels are filled with 50 mM aqueous KMnO4 solution (black trace).

Fig. 5 Measured absorbance as a function of concentration for a series

of aqueous solutions of KMnO4 over the concentration range from

100 nM to 100 mM.

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track the pH of a series of buffer solutions as they flowed

through the microfluidic chip. Fig. 6 shows the measured

absorbance as a function of pH over this range for the CRDS

measurements over the pH range 7.0 to 11.0. The measured

absorbance is directly proportional to the concentration of

deprotonated indicator, and follows the typical titration curve of

a weak acid, and the CRDS measurements are in good

agreement with single pass measurements made on a bulk

sample contained in a 1 cm cuvette.

For a known indicator concentration, the measured absor-

bance may be converted directly into a pH value using eqn (9).

Throughout the steepest part of the titration curve (pH range

y 9–10.5), the error on our experimental measurements allows

the pH to be measured to ¡ 0.028 of a pH point. Outside of this

range, the titration curve levels off and the absorbance is only

weakly dependent on pH, leading to much greater uncertainty.

These measurements demonstrate that provided an indicator can

be found whose colour change matches the pH region of interest,

and a probe wavelength identified that does not overlap with

absorption by the sample, introducing a low concentration of an

indicator compound to a sample and tracking its absorption

provides a general method for pH monitoring on a microfluidic

chip.

3.3 Oscillations of the Belousov–Zhabotinsky reaction

To demonstrate the potential use of CRDS for monitoring the

progress of chemical reactions occurring on microfluidic chips,

the Belousov–Zhabotinsky reaction was performed in the

thiolene-glass chip within the cavity, and the ringdown time

was monitored as a function of time from reaction initiation. The

reaction was carried out with a series of different concentrations

of the Ferroin indicator (3.125 6 1025 M to 5 6 1024 M) in

order to determine the optimum concentration for monitoring

the reaction both in CRDS and bulk absorption measurements.

Fig. 7 shows example data sets for an indicator concentration of

2.5 6 1024 M.

Under our reaction conditions, the oscillation period of the

reaction carried out on-chip is similar to that found in bulk

solution, around 70 s in both cases. However, the functional

forms of the oscillations in the absorption signal are quite

different for the two setups, indicating that the detailed kinetics

of the reaction are affected considerably by the shape and size of

the reaction vessel. After several oscillations, bubbles of CO2

(one of the reaction products) begin to form within the

microfluidic channels, degrading the ringdown signal; however

the bubbles do not appear to affect the period of oscillation.

These results demonstrate that CRDS measurements are easily

able to track the kinetics of reactions occurring on a timescale of

seconds. In principle, it should be possible to track events

occurring on the ms timescale, with even shorter timescales being

possible if a time-varying concentration is allowed when fitting

the ringdown traces. However, the time resolution of the

instrumentation used in this demonstration is limited by the

data transfer rate from the oscilloscope to around 250 ms.

3.4 Monitoring droplets in microfluidic systems

In our final demonstration, the CRDS setup was used to follow

the flow of droplets or ‘slugs’ of an aqueous phase seeded in a

toluene carrier phase, within a microfluidic channel. Fig. 8(a)

and 8(b) clearly show the change in ringdown time for two

different sets of flow conditions as the slugs of Rhodamine

solution pass through the laser beam. To acquire these data, the

toluene carrier phase was injected into the chip shown in Fig. 1

through port A at a flow rate of 0.003 mL min21 (Fig. 8(a)) or

Fig. 6 Absorption measurements as a function of pH for buffered

solutions of phenolphthalein indicator. Two different buffers were used

to cover the range from pH 7.0 to 11.0.

Fig. 7 Absorption signal recorded as a function of time for the

oscillating Belousov–Zhabotinsky reaction. The grey trace shows the

single-pass absorption recorded at 532 nm when the reaction was carried

out in a 1 cm cuvette, while the black trace shows the absorption

measured by CRDS when the reaction was carried out on the

microfluidic chip shown in Fig. 1(b).

Fig. 8 Droplet flow analysis using CRDS on a microfluidic chip. The

ringdown time t is tracked as droplets of 1 6 1026 M R6G solution in a

toluene carrier phase are flowed through a thiolene-glass chip at a total

flow rate of (a) 0.008 mL min21 and (b) 0.075 mL min21.

5382 | RSC Adv., 2012, 2, 5376–5384 This journal is � The Royal Society of Chemistry 2012

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0.05 mL min21 (Fig. 8(b)), and a 1026 M aqueous solution of

Rhodamine 6G was flowed through port B at 0.005 mL min21

(Fig. 8(a)) or 0.025 mL min21 (Fig. 8(b)).

The behaviour of t has several notable features. A clear

change in ringdown time is seen as the aqueous (droplet) and

toluene (carrier) phases pass through the laser beam. For the

data shown in Fig. 8(a), for example, the ringdown time switches

from y 220 ns for the toluene phase to y 260 ns for the aqueous

phase. As described earlier, the mounting angle of the chip

within the cavity has been optimised for measurements on

aqueous phases. The refractive indices of water (n = 1.333) and

toluene (n = 1.496) differ sufficiently that the ringdown time is

longer for the aqueous phase than for the toluene phase, even

though Rhodamine absorbs light at the wavelength of the laser.

For experiments in which quantitative absorption analysis of

droplets is the goal, the chip mounting angle should be optimised

to achieve the greatest difference in ringdown time between the

droplet and carrier phases. This was the case for the data shown

in Fig. 8(b), in which the ringdown times are y240 ns for the

toluene phase and y340 ns for the aqueous phase.

At low flow rates, a sharp reduction in t is observed as the

boundaries of each droplet pass through the laser beam. This is

caused by the meniscus at the droplet/carrier phase boundary

forming a lens that scatters the incident laser light out of the

cavity, dramatically reducing the ringdown time. The consistent

form of these scattering signals suggests that the topology of the

interface between the two immiscible solutions is identical for

each droplet, and a quantitative analysis of the signals could

even be used to study droplet morphology. This structure is

much less visible in Fig. 8(b), mostly due to the fact that the

droplet flow rate in this experiment was close to the limiting time

resolution imposed by our current fairly slow data acquisition

system.

These results demonstrate the ability of the two-mirror CRDS

system to interrogate and distinguish between individual

droplets. The acquisition time of the ringdown is of the order

of hundreds to thousands of nanoseconds. With an improved

data transfer system (currently limited at 4Hz), measurements at

kHz or even MHz data rates should become possible, allowing

the spatio-temporal resolution of fast moving droplets, com-

mensurate with kHz microfluidic label-free droplet production

systems.

4 Conclusions and future work

Thiolene-glass microfluidic chips have been integrated into a

two-mirror cavity ringdown spectrometer. By placing a chip at

an angle of 59u to minimise reflective and refractive losses, a

detection limit of 1.0 6 1023 cm21 has been achieved in a sample

volume of 132 nL, corresponding to a 214 nM minimum

detectable concentration of KMnO4. To illustrate the potential

of the system for monitoring a variety of different processes on

microfluidic chips, we have presented a number of proof-of-

concept demonstrations, including the measurement of pH,

reaction monitoring, and droplet flow monitoring. A number of

improvements are planned for the future, including minimising

the laser ‘footprint’ on the chip to allow smaller microfluidic

channels to be probed, expanding the method to probe at

multiple wavelengths or across a continuum of wavelengths,54

and upgrading the data acquisition system to allow measure-

ments at rates exceeding the kHz range.

Acknowledgements

This work was funded by the United Kingdom Engineering and

Physical Sciences Research Council through grant EP/G027838/

1.

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