Raman Micro Fluid i c Review

5880 Chem. Soc. Rev., 2013, 42, 5880--5906 This journal is c The Royal Society of Chemistry 2013

Cite this: Chem. Soc. Rev.,2013,42, 5880

Microfluidics and Raman microscopy: currentapplications and future challenges

Adam F. Chrimes,*a Khashayar Khoshmanesh,a Paul R. Stoddart,b Arnan Mitchella

and Kourosh Kalantar-zadeha

Raman microscopy systems are becoming increasingly widespread and accessible for characterising

chemical species. Microfluidic systems are also progressively finding their way into real world applications.

Therefore, it is anticipated that the integration of Raman systems with microfluidics will become

increasingly attractive and practical. This review aims to provide an overview of Raman microscopy-

microfluidics integrated systems for researchers who are actively interested in utilising these tools. The

fundamental principles and application strengths of Raman microscopy are discussed in the context of

microfluidics. Various configurations of microfluidics that incorporate Raman microscopy methods are

presented, with applications highlighted. Data analysis methods are discussed, with a focus on assisting

the interpretation of Raman-microfluidics data from complex samples. Finally, possible future directions of

Raman-microfluidic systems are presented.

1. Introduction

Microfluidics, which deals with geometrically constrained smallvolume fluids, allows for the flexible and highly controlledmanipulation of liquids, suspended particles and biologicalsamples. It is also well-known that Raman microscopy is apowerful tool that can provide unparalleled insight into the

a School of Electrical and Computer Engineering, RMIT University, 124 LaTrobe St,

Melbourne, Australia. E-mail: [email protected]; Fax: +61 3 9925 2007;Tel: +61 9925 3254

b Faculty of Engineering and Industrial Sciences, Swinburne University of

Technology, Glenferrie Rd, Hawthorn, Australia. E-mail: [email protected];

Fax: +61 3 9214 5160; Tel: +61 3 9214 5839

Adam F. Chrimes

Adam Chrimes graduated withBEng (Honors) in electricalengineering from RMIT University,Melbourne, Australia. He iscurrently undertaking a PhDdegree in nanotechnology enabledRaman-microfluidic systems atRMIT University, where he iscurrently receiving an AustralianPostgraduate Award to fund hiscandidature. Adam has authoredand co-authored more than 10scientific papers in peer-reviewedjournals and conference proceed-

ings during his three year PhD degree. His research interests includemicrofluidics, bio detection, Raman spectroscopy, electronics anddielectrophoresis.

Khashayar Khoshmanesh

Khashayar Khoshmanesh receiveda PhD degree in BiomechanicalEngineering from Deakin Univer-sity, Australia in 2010 with a focuson microfluidics and dielectro-phoresis. He has authored orco-authored more than 48 scientificpapers in peer-reviewed journalsand international conferenceproceedings. He was awardedthe 2012 American-AustralianAssociation Fellowship toconduct research at StanfordMicrofluidics Laboratory, Stanford

University, USA. As the recipient of the 20122015 Discovery EarlyCareer Researcher Award by the Australian Research Council, he is aResearch Fellow at RMIT University, Australia. His research interestsinclude manipulation of bio-particles in microfluidics undermechanical and electrical forces.

Received 17th December 2012

DOI: 10.1039/c3cs35515b

www.rsc.org/csr

Chem Soc Rev

REVIEW ARTICLE

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This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42, 5880--5906 5881

organic and inorganic chemical components and biomaterialsat low sample volumes. Combining Raman microscopy withmicrofluidics allows for the accurate monitoring, detection andanalysis of a wide range of samples in microfluidic environments.

The Raman phenomenon was first discovered by SirChandrasekhara V. Raman in the 1920s,1 and since then, progresshas been made in understanding the mechanisms and theoreticaldescriptions of the eect. Raman spectroscopy is particularlysuited for analytical chemistry, given that it is generally non-destructive, requires little or no sample preparation, oers highdiscrimination between sample components and is capable ofstudying gaseous, aqueous and solid samples.25

Raman microscopy is an advanced spectroscopic technique,incorporating optical microscopes, excitation lasers, opticalfiltering and manipulation devices, and spectrometers. TheRaman microscope oers the advantages of high spatial

resolution and optical sensitivity, due to the increased photonflux from the highly focused laser source and high collectioneciency of the objective lens. By providing specific informationabout the vibrational energy levels of chemical bonds and mole-cules, Raman microscopy is an invaluable tool for fingerprintingmaterials, and is finding increasing applications in medicine,biotechnology, material sciences and even forensics.69

The field of microfluidics emerged in the early 1980s and itis now routinely used in a variety of commercial applicationsincluding inkjet print-heads, lab-on-a-chip (LOC) systems,deoxyribonucleic acid (DNA) chips, and micro-thermal coolingdevices to name just a few. Of particular interest to this revieware the microfluidic systems that allow for precise manipula-tion of fluids that are geometrically constrained to smallvolumes, in the order of micro- and pico-litres.10,11

Microfluidic systems are becoming increasingly attractive inchemistry and biochemistry, as they allow for the miniaturisationof systems that are normally employed in those laboratories.Microfluidic systems are also useful tools for the handling offluids and suspended materials. As such, they improve theeciency of procedures by enhancing material mobility and vastlyreducing required sample volumes. Advances in microfluidicshave resulted in the creation of many innovative technologies inmolecular biology processes, proteomics and DNA analysis.1114

Microfluidic platforms that integrate optical and spectro-scopic analysis, exploiting both absorption and scatteringtechniques, have been extensively reported.15,16 Dierent typesof spectroscopy systems have been used with microfluidics,including ultraviolet visible absorption (UV-Vis), Fourier trans-form infrared spectroscopy (FTIR), fluorescence spectroscopyand Raman spectroscopy (inelastic scattering). Amongst thesetechniques, Raman spectroscopy has proven to be highlycompatible with microfluidics. Driven by improvements inhardware, the technique has largely overcome the concern thatit is a weak eect, as Raman scattering signal intensities are

Arnan Mitchell

Prof Arnan Mitchell (PhD 1999,RMIT) is Chief Investigator andRMIT Node Director of the ARCCentre of Excellence for ultrahighbandwidth devices for opticalsystems (CUDOS). He leads ateam of more than 30 members,spanning integrated optics,photonic signal processing,functional materials, micro-systems, nanomaterials and lab-on-a-chip technology. He and histeam focus on engineeringplatforms that enable funda-

mental scientific and biomedical breakthroughs and haveproduced over 200 publications in the past 10 years. Mitchell isalso committed to industrialisation of his research, holding anumber of patents and actively pursuing projects in defence,communications and biomedical diagnostics.

Kourosh Kalantar-zadeh

Kourosh Kalantar-zadeh is aProfessor at RMIT University,Australia. He received hisBachelor of Science. (1993) andMaster of Science (1997) degreesfrom Sharif University ofTechnology, Iran, and TehranUniversity, Iran, respectively,and his PhD at RMIT University,Australia (2001). His researchinterests include chemical andbiochemical sensors, nanotech-nology, microsystems, materialssciences, electronic circuits, and

microfluidics. He is the author of over 250 scientific manuscriptsand textbooks.

Paul R. Stoddart

Paul Stoddart graduated withBSc (Honours) in physics andPhD in laser spectroscopy fromthe University of theWitwatersrand, South Africa.After working on industry-focused surface science andmicroanalysis problems in anational lab for three years, hejoined Swinburne University ofTechnology in 2001. He iscurrently an Associate Professorin Biomedical Engineering atSwinburne. His current interests

include applied optics and biophotonics, with projects in the areasof fibre optic sensors, Raman spectroscopy and infrared nervestimulation.

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much smaller than those obtained by other spectroscopicmethods.17 When dealing with low volume samples, and possiblylow concentration analytes, Ramanmicroscopy provides sub-micronspatial resolution with very high sensitivity and selectivityfor microfluidic systems. Raman microscopy can also provideinformation regarding target materials very rapidly, withlatencies measured in the order of seconds, or even fractionsof a second, allowing for real-time monitoring processes to bepractically considered in such systems.

Integrated Raman microscopy and microfluidic systems(Raman-microfluidics) have already found a plethora ofapplications in the analysis of materials from low volume liquidmedia, especially when samples are rare and expensive(medical samples; forensic traces and pharmaceuticals), inmicroreactors for which constant monitoring is required(pre-processing of biochemical samples and tissue engineering)and monitoring of environmental samples (water quality andbiosensing). This track record suggests that the integration ofRaman microscopy as a powerful analytical tool, along with theunique properties of microfluidics, will be a key enabler formany future applications.

Many reports on Raman microscopy-microfluidics integra-tion exist. However, a comprehensive review on Raman micro-scopy in microfluidic environments is currently lacking. Thisreview aims to provide the reader with a detailed understandingof the capabilities of such systems and the opportunities andchallenges they present. The article has been designed to beinformative for a broad range of specialists, including those inthe fields of microfluidics, vibrational spectroscopy, optics,biotechnology, biochemistry, chemistry and analytical chemistrywho seek to exploit Raman-microfluidic systems for their diverseapplications.

2. Raman microscopy methods

To better understand the specific capabilities and limitationsof Raman microscopy-microfluidics, this section presents anoverview of the instrumentation and critical parameters, on anumber of variations on Raman microscopy, and then discussesthe opportunities and challenges of each of these in the contextof microfluidic devices.

2.1 Instrumentation and critical parameters

The Raman eect takes place when light illuminates a region,interacting with the molecules that are present in this region.The incident photons do not have sucient energy to excite aquantum transition from one stable electron configuration toanother, but the photon interaction does perturb the electronconfiguration of the molecule to an unstable virtual stateduring photon scattering. Most commonly, these scatteredphotons have the same energy, (and therefore frequency andwavelength) as that of the incident photon. However, a smallfraction of photons (on the order of 1 in 106) are scattered witha change in energy (and hence frequency). The dierence canbe attributed to energy gained or lost to vibrational energy inthe molecule. As such, the photon energy can be shifted to

lower or higher frequencies depending on whether they lose orgain energy. These shifts in frequency are called Stokes andanti-Stokes shifts, respectively. For molecules to exhibit theRaman eect they must have non-zero polarisability, that is tosay that an incident photon must be able to eectively deformthe electron configuration of the molecule. The degree of thisdeformation determines the Raman scattering intensity, due toresonant interactions with the rotational and vibrational statesof the molecule.

Raman-microfluidic systems can interrogate materialssuspended in liquid media or at the interfaces with liquids.Many types of liquids have distinct Raman signatures, allowingthem to be identified, as well as recognition of mixtures ofdierent liquid types. Solid suspensions and dissolved gasescan be dierentiated using Raman measurements in micro-fluidic environments. Additionally, Raman microscopy systemsare one of the best tools for understanding the properties ofsolid/liquid interfaces as the excitation beam can be tightlyfocused at the interface.

In Raman-microfluidic microscopy, a laser beam is focussedinto the microfluidic environment through the use of a microscopeobjective lens. The lens also collects the light, which is back-scattered from the sample, and passes it to the spectrometer viaa dichroic (color separating) filter. Before entering the spectro-meter, the strong elastically scattered Rayleigh wavelength (with thesame wavelength as the incident beam) is removed by the dichroicfilter, while the in-elastic Raman components are passed. Aconventional Raman microscopy system is shown in Fig. 1,which is comprised of a dichroic filter and a pin-hole with acontrollable diameter to ensure that only signals from the smallvolume at the focal point are collected. The spectrometer countsthe intensity of light collected at various frequencies.

Fig. 1 Schematic diagram of a confocal Raman system integrated with amicrofluidic unit.

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Raman spectroscopy allows for the integration with micro-scopic analysis techniques and is capable of collecting spectrafrom very small volumes (o1 ml), making it suitable for analysison the microfluidics size scale. Raman microscopy canapproach very high spatial resolutions. For example, using a633 nm laser source with a pinhole of 50 mm in radius and a60/1.2 numerical aperture (NA) objective, lateral and depthresolutions of approximately 0.25 and 1.7 mm, respectively, canbe achieved. These dimensions are comparable to features thatcan readily be realised in microfluidic systems.

The unit for Raman spectroscopy is the wavenumber, whichis the reciprocal of the wavelength shift relative to the laserwavelength, expressed as 1/Dl with units of cm1. Raman shiftsin the range 10400 cm1 can be used to study the rotationalaspect of molecular bonds, whereas the range 4004000 cm1

contains vibrations associated with vibrationalrotational struc-tures. For organic molecules the range from 5002000 cm1 isknown as the fingerprint region.

2.2 Variations of Raman spectroscopy

Raman spectra are inherently weak due to the rarity of theinelastic scattering phenomenon. Thus, much eort has beeninvested to design Raman systems with enhanced sensitivityand spectral resolution. Each enhancement technique comes atthe cost of some specific trade o. The following presents someof the key Raman spectroscopic variations and discusses theirrelative advantages and limitations for microfluidic devices.

2.2.1 Surface-enhanced Raman spectroscopy. Surface-enhanced Raman spectroscopy (SERS) was first observed byFleischmann et al.18 The eect originates from nanocolloids ofmetals, such as silver or gold, which generate localised surfaceplasmon resonances when excited by a laser source. Theseplasmonic eects can be observed in nanostructured surfacesof such metals as well. The existence of plasmon fields enhancethe intensity of the Raman signals from chemicals within theirrange, with enhancements as high as 108 reported for welloptimised systems.19 Although the fundamental electromagneticbasis of SERS is now well established, surface enhancement hasalso been associated with charge transfer eects in the metaladsorbate system (chemical enhancements).20,21 A large numberof excellent reviews, describing SERS and its principles are alreadyavailable.2124 Access to a variety of commercially availablecolloidal suspensions makes SERS suitable for microfluidicsand has been demonstrated for measuring many low concen-tration analytes such as 4-aminobenzenethiol,25 Rhodamine6G26 and crystal violet27 from inside microfluidic devices.While extraordinary enhancement, to the point of single mole-cule detection, has been demonstrated, it is crucial that theanalyte be in close proximity to and in the correct orientationrelative to the metal nanostructures, limiting the technique toanalysis of chemicals that can be eectively immobilised on thenanostructured metal surface. Whether that surface is a fixedstructure or nanocolloid does not change this requirement.Altogether, there are many examples which integrate microfluidicswith confocal SERS systems. Microfluidics are used in creatingsmall volume/high concentration samples for obtaining detailed

spectroscopic analytical information from the target materials.28

Microfluidics are used as to control the mixing between nano-metals and the target analytes prior to SERS measurements,29

and also provide solutions in controlling the spacing betweennanoparticles to enhance the SERS signal.30 For more informa-tion refer to reviews on SERS and microfluidics.31,32

2.2.2 Resonance Raman spectroscopy. Resonance Ramanspectroscopy (RRS) tunes the laser illumination wavelengthclose to one of the electronic transitions of the target molecule,leading to an enhancement of the Raman scattering intensityfrom the vibrational modes associated with the electronictransition, known as the FranckCondon active modes.33 Linesdue to other vibrational modes are also present in the Ramanspectrum, however these are not enhanced. This method iscapable of distinguishing Raman peak shifts of specific bondsin large organic molecules, such as chromophores,34 whichwould otherwise show complex Raman signatures in the spectralregion of interest.35 This method relies on tuneable lasers for theRaman excitation, which are readily available, but often withlimited power. Confocal techniques have been shown to directthe excitation beam onto specific locations of a target analytewithin a microfluidic system, thereby compensating for suchpower limitations.36

2.2.3 Surface-enhanced resonance Raman spectroscopy.Resonance Raman spectroscopy can be enhanced further throughcombination with SERS to form surface-enhanced resonanceRaman spectroscopy (SERRS). This combination allows the studyof specific bonds from extremely small sample volumes compatiblewith microfluidics dimensions.3739 SERRS can be readilyimplemented in microfluidic systems by introducing nano-structured metal surfaces or particles, enabling identificationand analysis of large proteins and other macro molecules suchas oligonucleotides37 and trinitrotoluene.40

2.2.4 Coherent anti-Stokes Raman spectroscopy. In Coherentanti-Stokes Raman spectroscopy (CARS), two pulsed laser beams,known as pump and probe beams, are used to generate anenhanced anti-Stokes photon. This method has been proven forspecific samples to be far more sensitive than traditionalRaman microscopy, and is gaining recognition in the scientificcommunity.4143 CARS microscopy has been particularly eec-tive in monitoring the structure and local environment of lipidsand water molecules, which may be useful for specialisedapplications in microfluidics.44 However, CARS is a non-linearoptical eect that relies on the exceptionally high peak powerpossible with ultra-short pulses. Dispersion must be carefullymanaged to maintain such high pulse powers and the pulseand probe must be carefully synchronised placing constraintson the geometry and stability of the optical interface to the micro-fluidic platform. Further, the intense pulses can cause multi-photonabsorption leading to strong fluorescence, or damage in materialssuch as polymers or organic matter. This constrains the choiceof materials for interrogation windows or analytes which can beprobed in microfluidic systems.

2.2.5 Stimulated Raman scattering. Stimulated Ramanscattering (SRS) microscopy is similar to CARS in that pumpand probe beams are used to make the molecular bonds


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oscillate in phase, while actively pumping the vibrational states,leading to significant enhancement of the Raman signal. Thekey dierence for SRS, as compared to CARS, is that the probebeam is at the Stokes wavelength. The intensity of the scatteredlight at the pump wavelength experiences a stimulated Ramanloss (SRL), while the intensity of the scattered light at the Stokeswavelength experiences a stimulated Raman gain (SRG). SRSmicroscopy has a major advantage over CARS in that it provideslow-background imaging with improved chemical contrast,45

both of which are potentially important for microfluidics wherewater is often the major source of non-resonant backgroundsignal in the sample. However, applications in microfluidicsmay once again be limited by dispersive broadening whenpassing through an optical window, limiting its applicationsin microfluidic systems for the time being.

2.2.6 Tip-enhanced Raman spectroscopy. Atomically sharpmetallic coated tips, such as those used in atomic force micro-scopy (AFM) machines, are used in tip-enhanced Raman spectro-scopy (TERS). When coated with nanostructured, plasmonicallyactive metals, these tips produce strong plasmon fields in theirvicinity. Hence, TERS can have the spatial resolution as small as10 nm, which has been demonstrated with single moleculesensitivity.4648 Furthermore, with the development of AFMs thatoperate in liquids, this technology has the potential to beincorporated into microfluidic devices for measurements at highspatial resolution.49

2.3 Considerations for Raman microscopy in microfluidics

Although Raman microscopy systems are relatively versatile, thereare still some constraints that must be considered, stemmingfrom the limitations of Raman microscopy and microfluidicsystems, as well as their integration. Specifically, these challengesrelate to the small volume of detection and how to manage thescattering process so as to optimise spectral collection eciency.It is also important to minimise damage to samples andmicrofluidic devices due to the optical power levels generatedby the Raman excitation systems. These issues are highlightedand discussed in more detail.

2.3.1 Droplet surfaces. When imaging droplets (frominside microfluidic channels, placed on substrates or on thesurface of open microchannels), the convex/concave shape of adroplet surface can adversely aect the Raman systems abilityto accurately measure materials suspended in such droplets.This is partly due to the fact that there is a refractive indexdierence between the droplet and the air. Additionally, theshape of the droplet surface creates a lensing eect that distortsthe focus and reduces the spatial resolution. Therefore, it isimportant to ensure that the Raman excitation enters thedroplet through as flat a surface as possible, ideally throughan optically transparent substrate, such as glass or quartz.50 Foropen microfluidic channel designs, it is important to considerthe width of the microchannel to ensure that the liquid is a flatas possible so as to minimise any issues caused by variousdistortions and chromatic aberration.51

2.3.2 Focal length. As the microfluidic device walls andsubstrates can be several millimetres thick, the focal length

must be long enough so as to penetrate inside the liquid media.Therefore the correct focal length is required to successfullyintegrate Raman microscopy and microfluidics. The focallength of a Raman system is determined by the optical arrange-ment of the microscope, but most importantly by the objectivelens. Lower magnification objective lenses tend to have longerfocal distances, generally making them more suitable formicrofluidic systems. High magnification, long working distanceobjectives can also be used, at the expense of optical intensity andhence, reduced signal to noise ratio. Water and oil immersionobjectives can also be considered. In some cases lenses are notnecessary, specifically in the case of fibre based Raman systems,where the fibre is integrated with the microchannel and the tip ofthe fibre exposed to the target liquids.52

2.3.3 Detection volume. Raman microscope systems focusthe excitation beam into a small volume using an objectivelens. The size of the detection area is dominated by the spotsize, or diameter, of the excitation beam at the focal point(Fig. 2). This diameter (d) is proportional to f the focal length ofthe lens and l the wavelength of the laser source and it isinversely proportional to D, the lens diameter (d p fl/D). Theother important parameter for the detection volume is thedepth of focus. The depth of focus (also known as the confocalparameter) is generally estimated as twice the Rayleigh range

(the distance between the

2p

d spot size points). The value can beapproximated as pd2/2l. In order to achieve a small depth of focusthe microscope must be operated in confocal mode, where thesize of the spectrometer entrance slit is reduced to the smallestvalue compatible with the required signal throughput.

To integrate Raman and microfluidic measurements, theoptimum spot size must be chosen, as the full focal volume isgenerally placed within the liquid medium to ensure that theRaman signals primarily arise from interactions with the targetanalytes. As mentioned previously, the use of high magnificationmicroscope objectives for Raman spectroscopy tends to increasethe signal to noise ratio and reduces the minimum detection limitby focusing the beam more tightly, and increasing the collectionangle. Targeted detection is also possible in microfluidics usingmechanical sample stages to move the targets with sub-micronspatial accuracy (e.g. piezoelectric stages).

Fig. 2 Schematic of the optical system showing the definitions of spot size,depth of field, focal length and detection volume.


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2.3.4 Excitation wavelength. The optimum choice of excita-tion laser wavelength is important for Raman spectroscopic appli-cations in microfluidics. The intensity of Raman scattering scalesinversely as the fourth power of the wavelength, so it is generallypreferable to use shorter excitation wavelengths. However, issuesarise when Raman microscopy of biological samples is required,because of the potential to produce large interfering fluorescencesignals as the photon energy increases. The shot noise of thefluorescence signal can mask the desired Raman signatures of thesamples.53 The majority of biological samples are also strongabsorbers of optical energy, particularly at shorter wavelengths(blue and UV). Modern Raman microscopes usually oer severaldierent options for excitation wavelength, however for thesesystems the diraction grating eciency and detector sensitivitymust also be taken into account for each operating wavelength.

For biological samples, the use of a typical excitation wave-length of 532 nm can produce such fluorescent signals. Onesolution is to use longer wavelength lasers such as red, or nearinfrared, so that the photon energy is below the fluorescenceexcitation band. While this may reduce the fluorescence in thesample, it also reduces the Raman scattering eciency of thesystem, requiring higher power lasers or longer integrationtimes. Additionally, the choice of excitation wavelength iscritical for resonance, and surface-enhanced resonance Ramanscattering. While resonant wavelengths may be advantageousfor detection at trace levels, other wavelengths may be preferredin situations where non-resonant bonds are of analyticalinterest.54,55

2.3.5 Optical power. In general, the scattering intensity canbe increased simply by increasing the excitation power, buthigh power densities at the focal point can cause damage tothermally liable biological samples in the microfluidic environ-ment. Additionally, high laser power at the focal point cancause localised turbulence or induce tweezing eects on nano-colloids in the microfluidics.56,57 Therefore, the optical powerfor Raman excitation should be carefully optimised in each case toensure that the sample is not damaged during the measurement.Much work has been done in determining the damage thresholdfor optical power applied to biological samples.57,58

Another strategy to reduce the potential damage to biologi-cal samples is to reduce the laser exposure time by loweringintegration times. However, as the integration time is reduced,the signal-to-noise ratio of the Raman spectrum is also reduced.The operator must determine a balance between biologicalmaterial damage and signal strength. The choice of the laserpower, together with the magnification of the objective lens,governs the power applied per unit volume of the analyte. Alarger spot size allows the use of larger powers. Generally, atmagnifications of 40 or higher it is better to keep the laserpower under 1 mW for large exposure times. This is a casewhere microfluidics are able to benefit Raman microscopy, asflowing fluids tend to dissipate heat energy and removedamaged analytes from the focal region, thereby allowinghigher laser powers to be used. However, the eventual eectdepends on the thermal conductivity of the medium and theparameters of the flow.59

2.3.6 Memory eect. Aside from the many benefits ofRaman-microfluidic integration, there are still a many challenges.One such challenge is the possible memory eect. This is due tosome particles and analytes sticking to the surface of the micro-channels, causing permanent Raman background signals.Many strategies can be devised to eliminate this problem. Forexample, disposable chips can be used to avoid such memoryeects. However, if reusable chips are desired then the wallsshould be carefully cleaned after each usage or they should beprotected during the process to avoid any target analyte inter-action with them. For instance, a segmented flow system can beimplemented, in which a thin layer of oil is used to protect themicrochannel walls from contamination.27,6062 There are alsomany other methods that can be applied to alter the hydro-phobicity of the walls and substrate, reducing the occurrence ofmemory eect issues.63

2.3.7 Portability. One of the challenges presented to theRaman-microfluidic community is the issue of portability.Microfluidic systems are highly portable, being small sized andrelatively robust. However the dimensions of Raman microscopyunits can bemuch larger, and particularly in the case of coherentRaman scattering, the alignment of the sample relative to theinstrumentation can be critical placing stringent constraints onthe optical interface to the microfluidic chip.

Low cost hand-held Raman spectrometers have spectralresolutions not exceeding 20 cm1 Raman shifts, while theresolution of laboratory spectrometers with optimised dirac-tion gratings, optical path lengths and cooled charged-coupleddetectors can be as small as 0.1 cm1. Similar concerns alsoapply to spatial resolution, while cost is a major determiningfactor for the sensitivity of the detector. Lower sensitivityspectrometers require higher laser power, which in turn limitsthe types of samples that can be analysed, particularly organicmaterials that can be damaged at high powers.

Eorts to reduce the size and complexity of the supportingoptical equipment are underway; covering ideas such as creatingon-chip laser sources64,65 and replacing the confocal lens withother devices such as waveguides66 and fibres.67,68 Recentlydesigned Kinoform microlenses for focussing into microfluidicchannels can potentially be used in Raman systems.69 Despitethese advances, the greatest diculty in achieving portability isreducing the size of the optical spectrometer while maintainingacceptable resolutions.

2.4 Materials

The type of material is an important consideration for systemsthat integrate Raman microscopy and microfluidics. From amicrofluidic perspective, it is important to know what materialsthe device is fabricated from, and what target materials are to beinterrogated. This is mainly to assure the least interferences fromunwanted materials and the largest signals from the targetanalytes to be obtained by the Raman system. This section aimsto highlight the various types of materials, and what considera-tions must be made for Raman-microfluidic devices.

2.4.1 Metals. Pure metals in bulk form do not produce anyRaman signature; this is due to the presence of free electrons in


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the metal structure, which block the incident light from reachingthe material bonds. This property makes metals reflective andgives them their high electrical conductivity.70 However, verythin films of pure metals have been shown to produce Ramansignatures, as the conductivity of nanometre thin films is lowenough to allow photons to reach the metallic lattice bonds.71

Nano-colloids and other roughened nanostructures are alsocapable of producing surface-enhanced Raman signatures, asdiscussed in the Variations of Raman spectroscopy section.The used of metal films or colloids to enhance Raman scatteringis particularly applicable to microfluidic devices.

2.4.2 Non-metals. Raman microscopy is capable of providinga good representation of inorganic, non-metallic materials;providing information regarding their chemical bonds, latticeand crystal arrangements. This capability is particularly usefulfor identifying dierent morphologies of materials, inorganicpolymorphs as well as dierentiation of amorphous and crystal-line material phases.70 Metallic oxides have unique Ramansignatures as seen in examples such as TiO2,

71 WO3,72 and

MoO3.7375 Additionally, Raman scattering can provide unique

information regarding the structure of materials like graphene,where it is possible to identify the number of layers and degreeof oxidation in its structure.76 Metal oxides can be used withmicrofluidic system as either trace analysis detection,29,62

interrogating the purity of samples,77 or the metal oxideparticles can be used to track non-active Raman elementsinside a microfluidic device.78 Non-metals such as glass, siliconand quartz are frequently incorporated in the structure ofmicrofluidics, which will be discussed in the Microfluidicconfiguration and requirements section.

2.4.3 Organic materials. Organic materials are importantin the context of constituents that form the microfluidicsdevices and the target material to be interrogated using themicrofluidic-Raman system. Changes in the character or quantityof particular organic bonds can be readily assessed by thelocation, strength and width of the Raman peak shifts. Asmentioned earlier, the Raman signatures provide detailedinformation regarding rotational and vibrational properties oforganic bonds. These Raman signatures can be well correlatedwith Fourier transform infrared (FTIR) signatures. FTIRspectroscopy is usually used together with Raman microscopyfor the analysis of organic materials. Although FTIR and Ramanspectra are similar, their signatures are not always identical.Polar organic molecular bonds, such as CO, NO and OH,produce relatively weak Raman signatures but strong FTIR

signatures. Conversely, neutral bonds such as CC, CH andCQC, are less easy to identify with FTIR, however they producestrong Raman peaks (Fig. 3). In the design of the microfluidics,care should be taken to avoid the interferences from theorganic material components of the microfluidic structure,which will be fully discussed in the Microfluidic configurationand requirements section.

Raman-microfluidic systems are great tools for providinghigh resolution and sensitive information regarding changesin cell metabolites and proteins, demonstrating distinct dierencesbetween cells at various stages of cancerous growth79 and candistinguish between healthy tissue, cancerous tissue and evenpre-cancerous tissue.80,81 Raman microscopy is also useful inmeasuring the type of polymeric materials in microfluidics, andtheir degree of polymerization. However, organic materials canbe damaged by high energy, low wavelength excitation sourcesas they expose the organic bonds to high energy photons.To accommodate this issue, Raman microscopy for organic,biological and medical specimens typically uses near infrared(NIR) lasers, such as 785 nm or 1064 nm, to reduce the photonenergy. This, however, reduces the intensity of the Ramanscattering process and necessitates highly sensitive Ramanspectrometers and longer integration times. Regardless, NIRRaman microscopy has successfully been implemented inmicrofluidics to classify epithelial pre-cancers and cancers.82,83

The ability of Raman microscopy to interrogate small detec-tion volumes can be used for the targeted analysis of largerorganic objects such as cells and tissues. Raman microscopycan be used for the identification of individual cells, whichhave been sorted and filtered using microfluidic components.The system can produce even more targeted information, withRaman signatures being taken from various parts of a largerobject, for example, targeting a cell where the Raman spectra ofthe nucleus, cell wall, cell membrane and cytoplasm can beacquired.2 This powerful option allows the detailed analysis ofa cells health, or the ability to monitor the absorption ofcertain chemical drugs.84 Another example has shown thatdierent locations on a yeast cell produce markedly dierentSERS spectra.85,86 Optical power density is again an importantconsideration when dealing with biological materials. Exposureto high optical powers has very detrimental eects on a cell, notonly thermally, but the high energy can irreversibly damage andeven kill a cell.57,87 For microfluidic systems the risk of damagefrom high energy optical systemsmust also be considered, as highoptical power can also aect the structure of the microfluidic

Fig. 3 Raman peak shift ranges for organic bonds.


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device, causing undesirable fluorescence and backgroundsignals to be introduced.

2.4.4 Aqueous media and microfluidics. Aqueous media arethe basis of microfluidics. Pure water produces weak Ramanpeaks at 1640 cm1 and at 3300 cm1 (Fig. 4) and can be readilyused for suspending organic materials, particles and analytes.8892

Water is also the main medium used to store and culture livingbio-materials. These benefits are what make Raman microscopyso powerful for microfluidics. The confocal ability of Ramanmicroscopy allows targeting specific areas inside an aqueoussuspension, with the ability to perform 3D Raman mapping.Furthermore, the intensity of the Raman peaks can be used todetermine the local concentrations of suspended materials inaddition to material identification as described previously.

Some fluids have strong Raman signatures, which can beused for their identification and assessing their concentrationin the microfluidics. Some of the examples of such measure-ments are presented in the Applications section. Examples ofRaman studies of aqueous media include alcohols, which showa strong presence of CH and CQC bonds and produce Ramanpeaks at 14001900 cm1. Additionally, Raman spectroscopyoperates not only on transparent samples but also on manysemi-opaque and opaque environments in microfluidics, forwhich many other optical systems fail.

3. Microfluidic configurations andrequirements

Integration of microfluidic and Raman microscopy systemsrequires many engineering steps. There are a number ofconsiderations for the integration of Raman microscopywith microfluidic systems, specifically relating to the designand fabrication of the microfluidic platforms. It is also impor-tant to highlight the potential benefits that microfluidic com-ponents such as mixers, filters and trappers can bring tointegrated systems.

3.1 Fabrication

Microfluidic systems are designed to deal with the behaviour,manipulation and precise control of fluids.93,94 Microchannelsare used to constrain the fluids to small scale areas, with somemicrochannels being less than 100 nm wide.95 This small sizeallows for the accurate control of flow, making such channelssuitable for the study of suspended materials and spatialmapping with high resolution. Microfluidics not only enable veryprecise flow control, but also has the potential for manipulation ofmaterials suspended in the liquid media by utilising mixing andseparating to optimise dierent chemical constituents.96,97 Micro-fluidic systems can easily be interfaced with standard laboratoryequipment such as syringe pumps, microscopes and electronicequipment to enhance the usefulness of their application.98

A further benefit of microfluidic devices is the ease offabrication. Soft lithography, a phrase coined by Whitesideset al.,99 is used to describe the processes for polydimethylsiloxane(PDMS) microfluidic device fabrication. Many other materials,such as glass, silicon and SU-8, can also be used for creatingmicrofluidic devices, each with a focus on a dierent applica-tion.101 As such, studies have been conducted to understandthe properties of these materials, their biocompatibility andtheir eects on analysis performed in microfluidics.102 Muchresearch has been conducted into microfluidic substratechoice and fabrication methods, with detailed analysis of suchmethods available in the literature.10,101,103107

Microfluidic devices based on rigid materials such as glassand silicon are often the basis of reusable systems and wheresevere chemical environments might be used in the micro-fluidics. However, the fabrication processes can be lengthy andrequire the use of high cost facilities and harsh chemicals.Conversely, PDMS is a relatively cheap material for the rapidfabrication of microfluidic devices. However, for PDMS basedmicrochannels there is still a need for a rigid structural plat-form. This substrate can be made from anything with solidstructural integrity, biocompatibility and preferably opticaltransparency. In fact, for Raman microscopy, a Raman trans-parent and low fluorescent material at the desired excitationwavelength is necessary.

Microfluidic devices can introduce significant Raman back-ground noise from the substrate and structures surroundingthe microchannels. Such background noise is more noticeableif the channels are made of polymers with strong Ramansignals (e.g. PDMS) and if the detection site is within closeproximity to the polymer, or the imaging must be done througha polymer membrane. This can be overcome by adding aRaman transparent window into the microdevice.40,108 Ramansignatures of various materials have been acquired using a532 nm laser source and are depicted in Fig. 4. PDMS andPerspex have many characteristic Raman peaks throughout theentire spectral range, making them unsuitable for use in the pathof the excitation laser. Silicon is usually used to ensure the correctalignment of Raman spectrometers, as it has a predictable andstrong Raman peak at 520 cm1. Glass has a very large andbroad Raman peak near 1000 cm1 making it unsuitable as a

Fig. 4 Normalised Raman spectra of possible microfluidic substrates such asPDMS, perspex, silicon, glass, quartz and water.


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microchannel substrate for detecting analytes with signaturesaround this range. Advantageously, quartz has relatively fewRaman peaks, with only a broad peak at around 350 cm1. As aresult, quartz is an excellent material as the substrate of choicefor Raman-microfluidics systems.

3.2 Optical transducers and environmental control

The measurement of parameters in microfluidics is performedusing transducers, which are required to operate with high sensi-tivity on small volumes of fluids. Examples of potential parametersto monitor include flow rate,109,110 viscosity,111 heat transfer,59

temperature,112 electrical impedance,113,114 permittivity,115

refractive index116 and other optical properties.117

Transducers can be integrated into microfluidic devices in avariety of ways: onto the surface of the substrates, on the wallsof the microchannels or integrated within the microfluidicstructures. They can be in direct contact with the fluids, orthey can be fabricated so as to enable contactless detection.Some examples of non-optical, non-contact methods of detec-tion that exist include capacitance sensors,113 piezoelectrictransducers118120 andmagnetoelectronic detection.121 Transducerscan be electrochemical, acoustic, thermal, electromagnetic andoptical.

The emphasis of this review is the integration of Ramanspectroscopy; however other transducers are in place to assistthis purpose. A good example for integrated optical detection isshown in Fig. 5(a), in which an optical fibre is used for Ramanspectroscopic detection of the analyte (in this case urea) in amicrofluidic channel.66 Similar fibre based Raman and SERSsystems with direct fluid contact have been proposed byothers.122 This method requires the optical fibre probes to bein direct contact with the fluid, exposing the fibre to possiblefouling and eventual damage. On the other hand, contactlessoptical detection in microfluidics can also be implemented, inwhich the laser is focused via an objective lens onto the area ofinterest for detection in microfluidics. Some recent examplesinclude the evaluation of microfluidic mixing using confocaloptical imaging to monitor the mixing of water and fluoresceindye123 and the detection of gas using a PDMS microchanneldesign integrated with confocal fluorescence imaging.124 Studies ofCO2 solubility in water (Fig. 5(b))

100 and metal oxide nanoparticleconcentrations108 are examples of Raman-microfluidic systemsfor online monitoring applications. A number of excellent reviewpapers summarise the many optical transduction methods usedin microfluidics.5,125,126

3.3 Considerations on flow, mixing, filtering and trapping

Careful control of the microfluidic environment is required tocapitalise on the high sensitivity and selectivity of Ramanmicroscopy systems. Regulation of the flow rate is critical, asintegration times for Raman spectroscopy are in the order ofmillisecond, seconds, and even minutes for some samples. Thelonger acquisition times require the targets to remain in thefocal area for the duration of the integration. An alternativewould be to implement microfluidic based traps to holdsamples in the Raman target area for the duration of the

acquisition time. These traps could be readily used for sortingpurposes also, as Raman microscopy information regarding thetarget can be used to control the sorting process.117 Furthermore,enhancement of the samples Raman signatures can be madeusing SERS, which would reduce the sample acquisition times.However, for many Raman-microfluidic systems, especially thosebased on SERS and for monitoring reactions (microreactors), itis necessary to ensure comprehensive filtering and mixing ofthe target materials, nanoparticles and analytes. This sectionwill highlight the considerations on flow, mixing, filtering andtrapping for Raman-microfluidic systems.

3.3.1 Flow systems and mixers. One advantage of micro-fluidic systems is the ability to provide continuous flow, whilestill using small volumes of samples. The integration of micro-fluidic flow cells with Raman analysis is important as it allowsfor the analysis of small volumes by positioning the sampleliquid into the Raman system detection area. Moreover, a higherreproducibility can be achieved in flow systems in comparison tostatic conditions due to the averaging eect over target material,and better heat dissipation of flow systems. Flow in microfluidicscan be driven by pressure or vacuum using external pumps.127

Other mechanisms also exist, such as capillary and electro-osmotic flows. The flow mechanisms in the microfluidic environ-ment have been comprehensively reviewed.128130

Mixing is an important aspect to be considered in Raman-microfluidics, as most microfluidic systems have low Reynolds

Fig. 5 Raman system integration with microfluidic environments. (a) Integra-tion of optical fibre detection into a microfluidic device for the purpose of in situRaman detection. Reproduced from ref. 66. (b) Confocal Raman microscopydemonstrating the detection of CO2 solubility in water. Reproduced from ref. 100(Copyright (2012), with permission from Elsevier).


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numbers and therefore intermixing is diusion limited.131 LowReynolds number systems are characterised by the absence ofturbulence, and the dominance of a laminar flow within thesystem. Examples of laminar flow are seen in many micro-fluidic structures, especially in T-junctions (Fig. 6(a)), where thefluids are not immediately mixed after entering the junctionand only intermix gradually through diusion.132 Raman beamrastering is one of the most ecient ways of assessingthe degree of diusive intermixing, which will be discussedfurther in the Applications section. Additionally, laminarflow systems can be manipulated and used for divertingstreams of fluids, as shown in Fig. 6(b)(e).133

As suggested in the introduction to this section, mixing is anessential part of many Raman-microfluidic systems especiallymicroreactors and SERS based systems. Microfluidic mixers areimplemented to enhance SERS signals, as mixing is vital forinitiating chemical reactions and introducing particles ornanostructures that enhance Raman through SERS. If liquidmixing is desired, then special systems must be implementedin order to induce disturbances and enhance the mixing ability.Microfluidic mixers are often used for accelerating the reactionof chemical constituents. Mixers can also be used for thecreation of precisely controlled nanoparticles, such as polymerbeads134 or metallic colloids.135 The mixing between a silver orgold colloid and a target analyte must be as comprehensive aspossible for obtaining high intensity SERS signals. Ramanmicroscopy is capable of detecting many liquid and solidcomponents from microfluidics, however it is also possible tomonitor mixtures of those components. Even more relevant isthe ability to use Raman microscopy for monitoring the mixingprocess itself, where Raman microscopy can provide informa-tion on desired and undesired chemicals produced duringmixing (and possibly a reaction progress) as well as giving anindication of the quality of output chemicals frommicromixers.However, from a microfluidic perspective, mixing only bydiusion is a time-consuming process, and relatively longmicrofluidic channel lengths are needed. A number of activeor passive micromixers have been developed to addressthis problem.137 Active mixers can achieve excellent mixing,but are comparatively dicult to integrate with other micro-fluidic components as they are relatively costly and generallyrequire complex control units and external power sources(e.g. bubble-induced actuators, magnetic stirrers, or ultrasonicwave generators). Passive micromixers are not as ecient andrapid but oer the advantage of easy integration with othermicrofluidic components, low cost and no external powersource.29,123 Examples of passive mixers are demonstrated inFig. 6. Micropillar microchannels for the mixing of silver colloidswith chemicals such as dipicolinic acid have been demonstrated(Fig. 6(f)).136 In a similar concept, alligator teeth-shaped PDMSchannels have been used for eective mixing of silver colloidwith DNA oligonucleotides (Fig. 6(g)).51

3.3.2 Traps, filters and sorters. Trapping, filtering andsorting of suspended materials can be implemented in amicrofluidic environment. For Raman detection and screeningapplications it is important to analyse specific bonds within

small and/or low concentration sample volumes. This requiresrelatively long acquisition times, therefore the target should beimmobilised for investigation. This is especially important inthe analysis of bio-cells in clinical experiments. Therefore,various trapping, sorting and filtering methods for delivery ofbio-components and cells to the detection area of Ramansystems and sorting of them with the use of microfluidic devices,are essential.138 All of these functions can be performed throughthe application of some type of force. The separation of materials,especially biological materials, is essential for processes wherepurer samples are desired. The active separation forces that canbe readily applied include optical, mechanical, electrokinetic,magnetic and acoustic (Fig. 6(h)(m)).117

In microfluidics, optical beams are commonly used fortrapping (also known as tweezing) suspended objects, allowinginterrogation of these particles using optical methods suchas fluorescence or Raman microscopy (Fig. 6(h)).139 Opticaltrapping can be performed using the Raman excitation laser ora separate laser beam using single or multiple objectives orfibres. In a recent example, optical traps have been used forisolating bacterial cells in liquid media.56 It is possible to thenchange the content of the fluid environment surrounding thecells by holding them in a trap while continuing to monitortheir response. Further to this approach, a waveguide opticaltrap was recently used on yeast cells.140 The waveguide trap hasthe advantage of causing minimal damage to the cells due tothe low optical power of the evanescent field. The use of opticaltraps is widespread for analysing cells in fluid environments,and many trap designs have been demonstrated.67,141148

In microfluidics, mechanical traps require no externalinputs beyond the fluid flow itself in order to operate, andcan also allow for the controlled release of the trappedobjects through manipulation of the flow. Mechanical trapsuse specifically designed barriers for holding objects inposition, and are usually implemented for long term studies,including studies of cells (Fig. 6(i)). If an object in such amechanical trap is to be interrogated using Raman microscopy,it has to be considered that the materials used in fabricatingsuch traps can also interfere with the Raman signals. Examplesof mechanical traps include those for holding single cells,where air bubbles are then used to eject the cells at the endof the measurement cycle.149 Other examples use mechanicaltraps for the optical imaging of live cells in microfluidics, whileallowing for the ability to modify the fluid environment withoutdisturbing the imaging process.150

Electrokinetics is a broad term that covers many phenomenathat occur in fluid environments, specifically involving thedouble-layer surrounding the suspended materials and theelectrical properties of both the media and materials. The moststudied concepts of these eects, implemented in microfluidicsfor separation and trapping, are dielectrophoresis (DEP) andelectrophoresis.117,151156 DEP is defined as the force exerted ona suspended dielectric particle in the presence of a non-uniformelectric field. The magnitude and direction of the force is relatedto the electric field intensity, particle radius, permittivity of theparticle and suspending fluid, as well as the conductivity of both


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the particle and suspending fluid (Fig. 6(j)). DEP can be usedfor trapping and sorting almost any type of suspended materialsranging from nanoparticles and carbon nanotubes157 to cells158

and DNA.159 DEP based cell traps have been used for quantify-ing the per-cell levels of lactic acid production160 as well asthe trapping of DNA with specific strand lengths.159,161

Fig. 6 Microfluidic methods demonstrating flow principles, mixers and traps. (a) Conceptual rendition of the simplest form of the T-section system, where two fluidinputs enter through channels at the bottom and slowly diuse over the length of the microchannel. Reprinted from ref. 132 (Copyright 2001 with permission fromElsevier). (b) Schematic drawing of tangential microchannels, where the channels can exchange fluid through the shaded area of contact. Laminar flow experimentswith the aspect ratio, A, of the contact areas as: (c) 100 160, A = 1.6; (d) 100 44, A = 0.44; (e) 400 25, A = 0.063. Adapted from ref. 133 (Copyright (2001)American Chemical Society). (f) Schematic illustration of a pillar array PDMS based microfluidic channel for the SERS detection of hazardous materials. The dashedrectangle denotes the Raman measurement area. Reproduced from ref. 136. (g) Schematics of alligator teeth-shaped micromixer. Reproduced from ref. 51. Schematicexamples of microfluidic traps using forces using: (h) optical (i) mechanical (j) dielectrophoretic (k) electrophoretic (l) acoustic and (m) magnetic forces.


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Sorting systems based on DEP have been used for sorting cervicalcells into healthy and tumorous types and can potentially be usedas an early detection device for cervical cancer.162 Other systemsdemonstrate the separation of bacteria and yeast cells based ontheir diering dielectric properties.157,158 DEP has been integratedinto Raman-microfluidics for the trapping and mapping ofnanoparticles such as WO3.

108 It has also been used forcontrolling the dynamic spacing between silver nanoparticlesfor optimising the SERS signals of biomaterials.30

In microfluidics, electrophoresis can be used to move suspendedparticles under the influence of uniform electric fields. Forparticles with a surface electric charge, the electrophoresisprocess is aected by surface adsorbed species, and as aresult the external electric field exerts a motive Coulombforce (Fig. 6(k)). An application of electrophoresis has beendemonstrated for the separation and detection of chemicals ina hybrid SERS nanocomposite device.164

There are many more methods for trapping and sortingobjects in microfluidics,165 including acoustic166 and magneto-phoretic processes,167 which are yet to be integrated with Ramansystems. Schematics diagrams illustrating these processes arepresented in (Fig. 6(k)(m)). Thermal procedures can also beconsidered.168 Interested readers are referred to comprehensivereports which dissect the various methods into two categories,active and passive, and proceed to analyse their resolution,throughput and eciency.169 It is also noteworthy to include afinal example reported by Lutza et al.170 who used Ramanspectroscopy to image the eddy concentration distribution forvarious acoustic oscillations in microfluidics.

4. Applications

As discussed previously, both microfluidics and Ramanspectroscopy are extremely useful methods in their own right.The integration of the two allows us to capitalise on all of thebenefits available to microfluidics, such as low analyte volume,tight control of the microfluidic environment and portability.Additionally, Raman microscopy provides detailed analysis ofthe target materials in microfluidics, including informationfrom suspended solids, liquid and dissolved gaseous samplesand their environments.

4.1 Investigation of analytes

One of the most important features of Raman spectroscopy isthat it provides information relevant to the quantity and typesof chemical bonds in the sample. The Raman laser beam can bereadily focused into a small volume of material within amicrofluidic system and allows analysis of only that very smallvolume. Raman-microfluidic devices also show great promisefor the detection of analytes in complex mixtures, as accuratemeasurements of dierent analytes can be simultaneouslyconducted.126

Many organic liquids can be eciently detected with Ramanmicroscopy as they have a strong Raman cross-section. Thereare many reported examples that have exploited this property.For instance, Fletcher et al.163 studied a T-shape microfluidic

device for the mixing of ethanol and acetic acid. Ramanmicroscopic mapping was used for detecting the two analytesat various mixing stages. The results show a very well controlledlaminar flow in the system. Fig. 7 depicts the Raman beamrasterised image of the T-channel, demonstrating the accuracy ofanalyte detection in a microfluidic environment. This method usestwo distinct Raman peaks for the detection of ethanol (882 cm1)and acetic acid (893 cm1), and can only be applied to thoseanalytes which have non-overlapping Raman peaks. There aremany more examples of such applications.132,171173

Microfluidic micro-reactors are used for the careful controland study of reactions. Raman microscopy can be incorporatedin such systems to investigate small volumes, determining thepresence of analytes and catalysts during the entire reactionprocess. Raman microscopy oers high-information content,in-line detection within microfluidic based micro-reactors. A verygood example is found in the work of Leung et al. who used acontinuous flowmicro-reactor with in-line confocal Ramanmicro-scopy to measure the concentration of output constituents.174 Intheir work, the catalytic oxidization of isopropyl alcohol intoacetone was monitored in real-time. They were able to use themicrofluidic environment to vary the input chemicals andcontrol the product conversion to precise levels.

Detection of specific analytes in a liquid is something whichis generally of interest for sensing, monitoring low concentra-tions of precious materials, and many other process controlapplications. When dealing with low concentration analytes itis important to have a good understanding of the target analyteand to assure that the parameters of the incorporated Ramansystem have been well tuned for the detection of those targets.For instance, Raman systems which use NIR excitation can beeciently used for detecting, and accurately measuring theconcentration of organic samples such as glucose, lactic acidand creatinine.126

Detecting low concentration analytes can be dicult usingconventional Raman systems; however using SERS, SERRS, andTERS, with either fixed nanostructures or colloidal suspensions,it is possible to increase the apparent Raman cross-section ofthe target analyte. For these systems, pre-processors, such asmixers (see section Considerations on flow, mixing, filtering

Fig. 7 AT-junction microfluidic channel approx. 200 mmwide for the purpose ofmixing ethanol and acetic acid. Insert images show rasterised Raman images ofacetic acid and ethanol using their respective Raman peaks. Adapted fromref. 163 (Copyright 2003 WileyLiss, Inc.).


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and trapping), are required to encourage the ecient bondingof target analytes onto the nanostructures that are used forenhancing the signals. Such systems have been used for sen-sing concentration levels in the part-per -million or even-billion levels of analytes such as cyanide, dipicolinic acidand malachite green (Fig. 8(a) and (b)).29,136 There are manygood review papers covering relevant topics in this field.21,23,175

Further enhancements of Raman signals can be achieved bycontrolling the spacing between the SERS colloid at the detec-tion area. It has been shown that colloid spacings in the rangeof 110 nm produce significant enhancement over randomlypositioned colloid.176 This control can be either static or dynamicin nature. Static methods use organic and/or inorganic spacers thatchemically attach onto the surface of colloids, which can be usedfor obtaining desired gaps between the particles.177,178 The maindrawback of such static systems is the lack of real-time tuningability and the interfering Raman signatures seen from the spacers.Achieving dynamic spacing, without colloid aggregation, can bemore challenging. One method for obtaining the optimum spacinghas been demonstrated in our own laboratory,30 where dielectro-phoretic forces were used to control the spacing of suspended silvercolloid to detect low concentrations of dipicolinic acid.

The application of fixed nanostructures for the SERSenhancement of analytes in a microfluidic channel is alsopresented for the detection of many organic samples such ascomponents of blood.179 One of the challenges associated withthese methods is ensuring enough analyte makes contact withthe nanostructures within the microfluidic flow. Physical forcessuch as electrophoretic force can be used for bringing the targetmaterials into the enhancement range of the nanostructures.

The use of similar techniques to detect airborne analytespresents a challenge, as these analytes must first be capturedeither by absorption onto solid substrates or suspended inwater, after which Raman analysis can occur. In order toaccommodate this, open channel microfluidics, where one sideof the channel is exposed to the air environment, have beenintegrated with colloidal SERS (Fig. 8(c)).25

4.2 Materials science nano/micro particles

The ability to identify and measure suspended materials is veryuseful with applications in assessing water contaminants andmicro-reactor outputs, determining the concentration of

mixtures and identifying unknown suspended materials. Raman-microfluidics can be employed for determining the quality ofsoil ingredients (after required pre-processing), chemical inter-actions, catalyst activities and corrosion eects.

Traditional methods of detection involve long preparationprocedures, such as drying samples and/or centrifuging liquidsamples to obtain more concentrated solutions. These procedurescan be accelerated in microfluidic devices, after which Ramanmicroscopy is capable of identifying many materials, deliveringtheir chemical fingerprint.180,181 A good example is demon-strated in the work of Chan et al.182 where CARS detection ofsuspended sub-micron sized polystyrene beads was shown.This concept was then expanded for the trapping of unilamellarvesicles, where the Raman analysis indicated peaks present at1440 cm1 due to the CH2 component in the lipids structure ofthe vesicles.

Many nanoparticles are synthesised and kept in colloidalforms, which are suitable for microfluidic processing. Ramanmicroscopy can provide invaluable non-invasive informationabout such colloidal systems: the type of suspended nano-particles can be identified, the colloid concentration can bedetermined using the intensity of Raman peaks and even thesize of nanoparticles can be measured from the Raman peakshifts and their widths.183 Raman microscopy can be ecientlyemployed for nanoparticles that generate surface plasmonresonances, such as gold and silver colloids, and can be usedto determine their size during synthesis.184 Raman-microfluidicsystems are well-suited to analyse the interaction of chemicals andorganic analytes on plasmonic nanoparticles.185 Additionally,Raman microscopy can be used to determine other nanoparticlessuch as carbon based particles (carbon nanotubes, carbon blackand graphene), metal oxides and chalcogenides using their strongRaman peaks.72,108,183,186 The concentration of nanoparticles atdierent areas of the microfluidics can be mapped and theirrelation to the dynamic forces of the flow can be obtained.108

One diculty with handling suspended materials in micro-fluidic environments is the memory eect. This eect is due tothe attachment of materials to the inside surfaces of themicrochannels, as discussed in the Considerations for Ramanmicroscopy in microfluidics section. This memory eect canintroduce unwanted Raman signals, giving false readings of concen-tration, and requiring the regular replacement of microdevices.

Fig. 8 Raman-microfluidic device applications for detecting suspended analytes using SERS colloids. (a) Detecting of cyanide using a microfluidic mixer. Reproducedfrom ref. 29. (b) Detection of dipicolinic acid using a micropillar mixer. Reproduced from ref. 136. (c) Detection of 4-ABT using an open microchannel design.Reproduced from ref. 25 (Copyright 2007 National Academy of Sciences, USA).


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In an eort to minimise these issues an oil/water interface iscreated on the inside surface of the microchannel to minimisethe chance of material attachment. Additionally, the flowsystem is arranged to form micro-bubbles, or nanodroplets,of liquid in order to minimise the time the microchannelsurface is exposed to potential fouling materials.27,60

In order to improve the magnitude of the acquired Ramansignals, it is common to increase the concentration of thesuspended material prior to analysis. Microfluidics providesthe perfect platform for this step, as pre-concentration can beconducted on chip. Many dierent types of forces can be usedfor this purpose, such as ultrasonic standing waves187 ordielectrophoresis.108

4.3 Analysing biological samples

Raman-microfluidic devices are ideal for analysing biologicalspecies, as they provide distinct fingerprints for each of thesamples and as such, have found many applications in biologyand biomedicine. They are attractive devices for non-invasivebiological studies as live samples can be analysed, they havehigh sensitivity to even molecular scale interactions and henceonly very small sample volumes are required. This sectionaddresses biological applications of Raman-microfluidicdevices and highlights their uses in specific studies.

4.3.1 Proteins. Proteins play important roles in manybiological functions such as cell signalling, immune responses,cell structuring, cell adhesion and cell life-cycles. They are sensitivebiological constituents requiring non-invasive and non-destructivemeasurement methods. Proteins are made up of chains of aminoacids, which produce strong Raman signatures, specific to the typeof amino acids. As a result, they can be dierentiated with a goodaccuracy using Raman microscopy.

Proteins such as enzymes and antibodies, as well as carriersand membrane proteins, have been studied extensively inmicrofluidics.12,58,189,190 Raman-microfluidic devices can beeciently utilised in the study of the behaviour of proteins,their functionalities and their responses under various stimuli.Microfluidics can also be used for purification of proteins fromother components of a cell and this entire process can becontinuously monitored using Raman microscopy.191 Enzymesand antibodies can be incorporated into microfluidics toestablish highly sensitive label-free and molecule-specificbiosensors based on Raman microscopy.192,193 Additionally,SERS, SERRS and TERS can be used for enhancing the detec-tion of suspended proteins. An example by Yang et al. involvesthe SERS detection of cytochrome c and lysozyme proteins froma suspension at various concentration levels.194 Additionally,the SERS detection of BSA has been demonstrated using goldcolloid, with detection limits as low as 100 pM.195

4.3.2 Deoxyribonucleic acid (DNA) and ribonucleic acid(RNA). DNA and RNA are the storage media for genetic infor-mation and are constructed from either ribose or deoxyribosesugars. The importance of nucleic acids is clear, and theiridentification and characterization is paramount for manyresearch fields. DNA samples are employed in many applica-tions including the recognition of genetic abnormalities and

also used heavily for forensic identification. Other uses for DNAinclude genetic engineering, bioinformatics, disease identifi-cation and biosensing.196200 DNA produces Raman spectro-scopic signatures and can be readily dispersed in aqueousmedia, making Raman microscopy well suited for detectionin microfluidic systems that process DNA. The Raman detectionof DNA is widespread through the use of SERS, as SERS providesthe ability to coat colloids in DNA friendly substances.201,202

The use of a microfluidic SERS mixer to detect single DNAoligonucleotides has been shown to accurately determine theirconcentrations.51 The simultaneous detection of multiple DNAoligonucleotides using SERS has also been demonstrated,where a multi-gradient microfluidic channel was used forcontrolling the microfluidic environment. Labelling the DNAoligonucleotides can also assist in dierentiating between theRaman signals (Fig. 9).188

For DNA monitoring in microfluidics, many issues shouldbe taken into consideration. Polymerase chain reaction systemscan be integrated into the Raman-microfluidic device, provid-ing enough DNA strands for the detection. The application oflinkers, dyes and surface seeking groups are important factorsas they allow for the ecient adhesion and immobilisation ofthe DNA strands for monitoring processes.37,38 Pre-processingprocedures such as mixing and filtering are also important inDNA Raman-microfluidics. As an alternative to colloid mixing,there are reported implementations that use solid microchannelfeatures to achieve mixing and Raman enhancement.203,204

Fig. 9 Raman-microfluidic devices for the mixing and detection of two breastcancer-related DNA types (DNA1: 50-CTG TTT GCT TTT ATT-30; DNA2: 50-GCT GTTTAT TTA TTA-30). Raman spectra of mixtures with ratios of DNA1 to DNA2: (a) 0 : 1(b) 1 : 3 (c) 1 : 2 (d) 1 : 1 (e) 2 : 1 (f) 3 : 1 (g) 1 : 0. Reproduced from ref. 188.


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4.3.3 Cells. The structure of a cell is complex, with manybiological constituents in its make-up, such as proteins, DNA,amino acids and various other components. Ramanmicroscopyis well suited to the simultaneous detection and selectiveanalysis of the components of such complex mixtures. Addi-tionally, cells are more active in liquid environments, wherenutrients can be absorbed from the liquid, and communicationchemicals released.205,206 Microfluidic systems are ideal forsustaining cells in an aqueous environment where constantcontrol of the chemical makeup and other environmentalfactors is possible.205,206 Such control is especially importantfor extended studies where cells are cultured through theirentire life-cycle. Furthermore, Raman microscopy can be usedto monitor the chemical signatures of each stage of this lifecycle and hence allows for the classification of cells intolifecycle states, including viable, necrotic and apoptotic.207209

Raman-microfluidic systems are capable of identifying andstudying contaminating cells, including bacteria, for situationssuch as water quality control and studying the eects ofantibiotics on particular strains. A practical example of howRaman-microfluidic devices are used in such applications isdemonstrated by Knauer et al.28 A water quality monitor wasdeveloped in order to ensure that no E. coli cells were presentper 100 ml of water, and was designed to have internal antibodycapture sites for E. coli. (Fig. 10(a)). The system operationrequired a specific volume of water to pass through a micro-fluidic channel, fitted with E. coli trapping sites, after whichsilver colloid was introduced to coat any trapped E. coli cells.Using the prominent 565 cm1 Raman peak, the system wascapable of detecting the presence of any E. coli cells, even downto the single cell level.

It is possible to take advantage of the high confocality oered byRaman microscopic systems for the analysis of individual compo-nents of cells, avoiding the need to destroy the cell. As an example,Raman analysis has been performed on suspended Bacillus anthracisspores to determine the amount of calcium dipicolinate containedinside the spores.210 It is also possible to classify tumour cellsdepending on their internal chemicals, demonstrating howRaman-microfluidic devices can be used to identify specific typesof tumour cells from a larger sample of unknown cells.67

Microfluidic systems can be implemented to filter and sortcells, with the ability to create highly pure cell samples. Ramanmicroscopy is able to detect dierences and defects in cells, andwhen coupled with suitable microfluidics, can be used as thesensor component of a cell sorting system. An optical trap hasbeen used for such sorting, where lymphocytes were identifiedand sorted. Lymphocytes are vital to the human immunesystem and there are many variants with a range of specificfunctions.138 The cells are trapped at a Raman detection sitewhere they are then categorised and positioned inside specialholding areas, creating pure sample batches of the lymphocytetypes (Fig. 10(b)). Microfluidic traps provide Raman-microfluidicsystems with the capacity to study cells that are exposed to changingenvironmental conditions, with systems being used for studying theeects of medical drugs on cells. To this end, a Raman-microfluidicsystem to study the behaviour of yeast cells under various environ-mental conditions has been demonstrated.211

Temporal studies of cells are possible with Raman-micro-fluidic systems. As an example, Chinese hamster ovary (CHO)cells have been studied using a Y-shaped microfluidic channelused to mix cells with SERS colloid.212 The cells were monitoredover time using Raman microscopy, and it was found thatAmide I levels reduced over time. Furthermore, spatial experi-ments were also conducted on the CHO cells, creating an xyRaman map, together with a z-axis profile showed that therewas a strong indication of CH deformation peaks fromproteins near the centre of the nucleus.

4.3.4 Tissue. Raman-microfluidics has found applicationsin tissue engineering, where the benefits of such systemsinclude the continuous flow of nutrients and vital gases like oxygen,as well as the constant withdrawal of waste products.213,214

Additionally, microfluidics provides the perfect platform forstudying fundamental biological phenomena, including exposingtissue to environmental variations at critical growth stages tomonitor their eects.215 The applications of this technology areparticularly important for the study of drugs, where tests can beimplemented safely on live human tissue samples in a highlycontrolled environment while allowing for accurate measure-ments. This could drastically reduce the failure rate of potentialdrugs involved in human clinical trials.216 Some examplesextend this concept as far as organ-on-a-chip microsystems,which allow for in vitro organ-level studies, as opposed to cell-based systems.217 Raman microscopy can be applied to theseorgan-on-a-chip systems for in-line detection and monitoringof samples, providing accurate measurements of chemicalconstituents. Raman microscopy allows for non-invasive andnon-contact detection in the LOC environment, providing

Fig. 10 Applications of Raman-microfluidic systems for cell detection and sort-ing. (a) Schematic of a Raman-microfluidic device fitted with antibody capturesites. Cells are captured at these sites and are interrogated by Raman micro-spectroscopy. Image not to scale. Reproduced from ref. 28 (with kind permissionfrom Springer Science and Business Media). (b) Operating procedure andassociated images for a Raman-microfluidic cell sorter using optical trappingand manipulation. Reproduced from ref. 138.


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contamination-free and well controlled results. With the use ofSORS or transmission Raman it is possible to analyse 3D tissuesamples, with penetration below the surface of the tissuesamples, proving valuable feedback on the internal process oftissue samples.3,218223

4.3.5 Other organic samples. In addition to the aforementionedorganic samples, Raman-microfluidic systems are capable ofstudying many other biological candidates, including DNAcell,DNAprotein, virus, toxins and cellprotein complexes,224 andtheir compositions with inorganic chemicals.200

Raman microfluidic systems are also excellent tools forinvestigating zygotes and embryos. The monitoring of zygotesand embryos in microfluidic environments that simulate theirhost are particularly important for the investigation of geneticdeformations, abnormalities and related issues and provideinvaluable information about the process of their growth.Raman microscopy has already been used to identify and studyyeast zygote cells in an eort to apply such Raman mapping forthe label free study of cell proliferation.250,251

In vitro fertilisation (IVF) treatments currently rely on visualinspection of the morphology of an embryo in order to determineits viability as an IVF candidate. Research currently suggests thatRaman microscopy can assist in the identification of ideal candi-dates, in combination with morphology characteristics,252,253 withthe potential to increase IVF success rates.252,253 Furthermore,microfluidic environments can be used to enhance themonitoring and handling of embryonic cells, as microfluidicswould allow for high throughput approaches.254256 The micro-fluidic environment can even be used for the fertilisation ofembryos, allowing the initial stages to be carefully monitoredbefore transferring the embryo to the host.254,255,257

4.4 Pharmaceuticals

Ramanmicroscopy can be used for investigating the eectiveness ofdeveloped drugs in the controlled microfluidic environment. Dier-ent scenarios can be simulated and tested using Raman-micro-fluidic systems, using low cost processes and small volume samples,before the pharmaceuticals are tested on animals and human.Multi-component pharmaceuticals can also be tested and the resultscategorised using multivariate data analysis procedures.258

Microfluidic micro-reactors can be used for pharmaceuticalproduction and testing, and every stage of the process can becarefully controlled. Raman microscopy can be used to givereal-time feedback on the chemical reactions in such sys-tems.259 For example, Raman microscopy can support thescreening of polymorphic structures, and support the chemicaldevelopment process as it is scaled-up.261 Lipids are oftenessential components of colloidal pharmaceutics. Ramanmicroscopy has proven particularly sensitive to lipids, whichcontain unique Raman signal producing hydrocarbon chains.Raman microscopy can determine the structure of lipids todetermine the packing behaviour and phase transitions.262

4.5 Forensics

Accurate forensic analysis is required by authorities for obtainingsuccessful prosecutions. Current systems rely on electrophoretic

DNA chips, which require pre-processing, filtering and poly-merase chain reactions to enhance the number of DNAstrands.263 Alternatives to these DNA chips include opticaloptions such as fluorescent systems, which require the use offluorescent tags.264,265 Raman systems can also be used forDNA characterisation, providing a quick and thorough analysisof biological forensic markers. Raman analysis has been usedfor the forensic analysis of body fluid traces266 including wholeblood,267 vaginal fluids,268 forensic pharmaceutical investiga-tions,269 explosives6 and drugs of abuse.270

Microfluidics has the potential to enhance this field, as themajority of forensic samples are organic in nature and canbe suspended in liquid, allowing them to be measured inRaman-microfluidic devices. Furthermore, microfluidicsprovide ideal lab-on-a-chip environments, therefore samplepreparation can be conducted in a fast and portable fashion.Samples of DNA and other body fluids can be stored intheir liquid forms, and Raman microscopy provides a non-destructive method of testing, allowing the sample to bere-used if required. Despite all of these benefits there are stillthe issues of portability and cost, which have been thoroughlydiscussed in the Considerations for Raman spectroscopy inmicrofluidics section. Low cost devices and access to the rightdata banks are required for widespread forensic analysis usingRaman-microfluidic devices.

5. Data analysis

Analysis of data from Raman microscopy-microfluidic measure-ments is extremely important for gaining the correct insightsregarding the samples under investigation. Raman spectro-scopy data obtained from a microfluidic system is not asdefinitive as the information obtained

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Raman Micro Fluid i c Review

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