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Contact Lens & Anterior Eye 35 (2012) 137– 144

Contents lists available at SciVerse ScienceDirect

Contact Lens & Anterior Eye

jou rn al h om epa ge: www.elsev ier .com/ locate /c lae

ibre optics sensors in tear electrolyte analysis: Towards a novel point of careotassium sensor

aniel Harveya,∗, Neil W. Hayesb,1, Brian Tighea

Biomaterials Research Unit, School of Engineering and Applied Science, Aston University, UKEvanesCo Ltd, Forde Court, Forde Road, Newton Abbot, Devon, UK

r t i c l e i n f o

eywords:ibre optic Sensorear assayear analytesry eye

a b s t r a c t

The diagnosis of ocular disease is increasingly important in optometric practice and there is a need for costeffective point of care assays to assist in that. Although tears are a potentially valuable source of diagnosticinformation difficulties associated with sample collection and limited sample size together with samplestorage and transport have proved major limitations. Progressive developments in electronics and fibreoptics together with innovation in sensing technology mean that the construction of inexpensive point ofcare fibre optic sensing devices is now possible. Tear electrolytes are an obvious family of target analytes,not least to complement the availability of devices that make the routine measurement of tear osmolarity

possible in the clinic. In this paper we describe the design, fabrication and calibration of a fibre-optic basedelectrolyte sensor for the quantification of potassium in tears using the ex vivo contact lens as the samplesource. The technology is generic and the same principles can be used in the development of calciumand magnesium sensors. An important objective of this sensor technology development is to provideinformation at the point of routine optometric examination, which would provide supportive evidence

Britis

of tear abnormality.

© 2012

. Introduction

Point of care assays are increasingly important and rely on self-ontained, user-friendly desktop systems that can be used in thelinical environment. They desirably combine rapid diagnosis withinimum handling of the patient’s sample, thereby reducing time,

ost, and the potential for errors. The need for rapid analysis islearest in the case of critically ill patients; and potential ways ofmproving sensitivity, speed and versatility of point of care bio-hemical assays represent a key aspect of technique development1].

The availability of an increased range of point of care assays willreate new opportunities for biochemical screening to be used as andjunct to certain types of routine assessment – such as visits to anptometric clinic. In such situations the technology must be robust,ompact and suitable for use in a clinical, rather than analytical set-

ing. In environments other than the hospital clinic, non-invasivelternatives to blood samples – such as saliva or urine – are alreadynder active investigation. The use of tears as an assay medium has

∗ Corresponding author at: Biomaterials Research Unit, School of Engineering andpplied Science, Aston University, Birmingham B4 7ET, UK. Tel.: +44 0121 204 3390.

E-mail addresses: biostuff@aston.ac.uk, danharvs@hotmail.com (D. Harvey).1 Present address: Research and Knowledge Transfer, Innovation Centre, Univer-

ity of Exeter, Rennes Drive, Exeter EX4 4RN, UK.

367-0484/$ – see front matter © 2012 British Contact Lens Association. Published by Elsoi:10.1016/j.clae.2012.02.004

h Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

been less actively pursued, largely because samples are difficult tocollect reproducibly and resultant sample volumes are very low.This paper identifies the potential use of lens-borne tears in com-bination with emergent fibre-optic technology as a potential routeto a new area of point of care diagnostics in optometric practice.

The potential advantages of the use of tears as a means of obtain-ing analytical data relating to blood constituents was recognisedmany years ago [2]. Plasma leakage means that tear constituentsreflect many of those found in the blood supply to the brain, becausethe palpebral conjunctiva is supplied by the ophthalmic artery, abranch of the internal carotid artery, a major supplier of the brain.This process is responsible for the presence of albumin in the tearfilm [3].

A major problem exists, however, in obtaining tear sam-ples of adequate volume to enable measurement or detection ofconstituents by conventional analytical techniques. Attempts toovercome this problem by use of artificial stimulation of tear pro-duction are confounded by the fact that the concentration of sometear components is flow-dependent [4]. The method used for tearsample collection has, in consequence, a significant influence on theanalytical data obtained. Khuri proposed the use of Schirmer stripsas a means of overcoming this problem and demonstrated that this

method of collection does allow determination of organic compo-nents such as glucose and urea together with electrolytes such aspotassium and calcium [2]. Although the principle of this collectionmethod is sound and readily adaptable to point of care analysis

evier Ltd. All rights reserved.

138 D. Harvey et al. / Contact Lens & Anterior Eye 35 (2012) 137– 144

Table 1Collected literature values of osmolarity in comparison to clinical assessment for dry eye cases of varying severity.

Severity of dry eye Recorded osmolarity(mOsm/l)

Recorded osmolarity(mOsm/l)

Recorded osmolarity(mOsm/l)

Difference between highestand lowest recorded values(mOsm/l)

Normal 302.2 [7] 269.5 ± 9.8 [9] 302.2 ± 8.5 [10] 32.7

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Mild 298.1 [9]

Moderate 306.7 ± 9.5 [9Severe 326.9 [7] 314.4 ± 10.1 [

t has not been matched by a simple and sensitive assay tech-ique that would enable one or more important tear constituentso be measured conveniently and quantitatively. Additionally it haseen shown that this type of invasive technique, including surgi-al sponges and filter discs, causes irritation and leads to elevatedalues of some serum-derived tear components [5].

The last fifteen years has seen little advance in point ofare assay of specific tear analytes. Perhaps the most impor-ant recent development of this type has been the TearlabTM

ano-osmometer introduced commercially in mid 2009 [6]. Thisnstrument essentially measures total tear electrolyte concentra-ion [7]. The potential importance of this approach derives directlyrom the work of Gilbard who pointed out that tear film osmo-arity is potentially raised by any condition that increases tearvaporation or decreases tear secretion [8]. His studies translatedhe theoretical link between keratoconjunctivitis sicca (KCS) andear film osmolarity to observations with practical clinical utility,mphasising the importance of the link between tear electrolytealance and concentration on one hand, and the health of the ocularurface on the other [8].

It is clear that total tear electrolyte concentration (i.e. osmolar-ty) has important clinical significance, and that it has only beenhe lack of an accurate and affordable point of care assay that hasrevented its widespread use in optometric practice. In dry eye,

ncreased evaporation of the tear film leads to an increase in thesmolarity of the tear film. Because tear osmolarity is influencedy all tear electrolytes, its diagnostic significance is limited. Addi-ionally, point of care experience with the commercially availableearlabTM osmometer has been mixed [7,9,10]. This is illustratedy the values shown in Table 1, which collects together reportedear osmolarities and associated clinical assessments for a range ofry eye patients.

For a clinical sensor system to be of maximum value it musthow both accuracy and reproducibility in assessing specific clinicalonditions. The results in Table 1 show good general correlation inifferentiating between dry eye conditions of varying severity, butlso highlight a considerable degree of site-to-site variation in thepparent clinical significance of absolute tear osmolarity values.

Khanal et al. reported that the use of TearlabTM requiredhree consecutive readings to obtain an average reading with 95%onfidence, whereas in a clinical diagnostic scenario ideally oneeasurement should suffice [11]. The readings obtained with clin-

cal samples were found to vary by up to 35 mOsm/l for threeonsecutive readings one minute apart, and with nineteen repeatedeasurements of the standard solution (290 mOsm/l) by up to

9 mOsm/l [11]. These observations support the view that point ofare diagnostics involving tear samples are still in an early stage ofevelopment. The observed lack of absolute consistency reported

n the literature is of some concern, considering that osmolarityeasurements are used as a diagnostic tool. Arguably, additionalork is needed in the assessment of tear electrolyte levels and in

he development of tear sample collection techniques. These points

re addressed by the present work; in particular the merits of indi-idual tear cation assays. Potassium is a useful initial target analyte.

Tears contain between 15 and 30 mEq/l (milli equivalents peritre) of potassium ions, a much higher than the level found in

315.0 ± 11.4 [10] 16.9315.0 ± 11.4 [10] 8.3336.4 ± 22.3 [10] 22

serum, which is around 4.5 mEq/l, indicating that there is activepotassium secretion in tears [12]. Concentrations are often quotedin units of mmol/l or mM rather than mEq/l. For the potassiumion K+, which has a valence of 1, these units are numerically equalwhereas for divalent ions (ions with a 2+ charge) such as calcium(Ca2+) or magnesium (Mg2+) 1 mmol/l or 1 mM is equal to 2 mEq/l.Tear calcium and magnesium levels are closer to those of intersti-tial fluids than those found in plasma. Interstitial fluid calcium andmagnesium are critical for a variety of neuromuscular functionsand low levels can cause a range of abnormalities in behaviour,including convulsions. Typical concentrations of ionic calcium intears are around 0.4–0.8 mM, as compared to 1.09–1.33 mM inplasma. Magnesium ion levels in tears are 0.5–1.1 mM, as comparedto 4.36–5.32 mM in plasma) [13–15].

The lachrymal gland is the major source of tear electrolytesalthough their concentration in the tear film, as Gilbard demon-strated, are markedly affected by evaporative loss and consequentdry eye [16]. A secondary source of tear components arises becauseof plasma leakage, a process that makes the use of tears poten-tially interesting for the determination of serum levels of lowmolecular weight species such as glucose [17–19]. Since albu-min, with a molecular weight of some 80 KDa, diffuses into thetear film by vascular leakage [3] it is logical to expect thatmuch lower molecular weight species such as electrolytes willenter the tear film by this route quite freely [3]. It might there-fore it would be expected that the osmolarity of blood serumwould be similar to that of the tears. In fact the normal reportedrange of blood serum is 275–295 mOsm/kg or 291.5–312.7 mOsm/l[3,20,21], compared to the literature reported range of normal tearswhich is 269.5–302.2 mOsm/l [7,9,10].

Serum potassium levels are clinically significant and prob-lems related to potassium concentration are often detected duringscreening blood tests for a medical disorder. In less fortunate casesthe condition only comes to medical attention after complicationshave developed. Perhaps surprisingly, potassium disorders are sec-ond only to hydrogen ion disorders as causes of mortality andmorbidity [23].

Potassium-related abnormalities range from hyperkalemia (inwhich the serum potassium ion concentration is greater than5.5 mM/l) to hypokalemia (in which the serum potassium ionconcentration is less than 3.5 mM/l). Hyperkalemia is the more dan-gerous and can lead to fatal cardiac arrhythmias [24]. Hypokalemiacan be caused either by inadequate potassium salt intake, excessivepotassium ion renal excretion or gastrointestinal loss. Though themeasurement of serum potassium via conventional blood tests hassaved many lives, many cases are missed because of the lack of anon-invasive, monitoring system [23].

The other potential sources of potassium in tears are cornealand conjunctival cells, but the possibility is apparent rather thanreal. There have been many experimental and modelling studies onthe transport of ions in the human cornea and the correspondinginfluences on corneal hydration. Under normal in vivo physiologi-

cal conditions, there is no net fluid transport, since the movementof fluid into the cornea driven by the stromal swelling pressureis exactly counterbalanced by the active secretion of fluid outof the cornea [25]. This ‘pump-leak’ mechanism hypothesis for

D. Harvey et al. / Contact Lens & Anterior Eye 35 (2012) 137– 144 139

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The underlying principle is simple: a light beam from an LED(light emitting diode) is passed through a fibre optic cable andreturned to a detector which can detect any change in signal.At a point along the fibre a sensor coating that incorporates a

ig. 1. The total sensor system showing the EvanesCo EV5000 light source and deens. The EV5000 box is connected to the laptop controller.

aintenance of corneal hydration and transparency has existedor several decades since the hypothesis was first made [25]. Itas been suggested that the endothelial pump leak involves com-licated ionic exchanges (such as Na+/H+ and Cl−/HCO3

−) andon cotransport (such as Na+/HCO3

− and K+/HCO3−, Na+/K+/2Cl−)

26,27]. Because the volumes of tears and aqueous humour areuch greater than the stromal volume it is generally assumed that

he concentrations of dominant ionic species found in tears and inqueous humour (Na+, K+, Cl− and HCO3

−) are not affected by elec-rolyte and fluid transport processes in the epithelia of the corneand conjunctiva [26–31,25].

The value of analytical screening procedures, particularly atoint of care has been frequently, made but in the case of tearsample collection and assay techniques present particular prob-ems. It is clear that in comparison with general observations ofear osmolarity, elevated levels of specific electrolytes are associ-ted with specific conditions, and thus offer diagnostic potential.or this reason elevated potassium levels were recognised manyears ago as a potential early marker of dry eye [33]. Similarly, ele-ated calcium levels are not only a consequence of meibomiumland dysfunction, but have also been shown to be a key charac-eristic of primary acquired nasolacrimal duct obstruction [33,34].n a more trivial level, it has been suggested that elevated tear cal-ium levels predispose patients to form white spots during contactens wear. The availability of a suitable point of care assay wouldnable the general applicability of such propositions to be readilyetermined and additionally aid practitioners in materials selection35].

Point of care measurement of osmolarity is becoming moreidespread and enhancing understanding of the clinical advan-

ages and limitations of the technique. The extension of clinicaleasurement capabilities to include specific electrolyte compo-

ents is a logical step. The technique described in this paper isnique in its approach because it uses existing transferable tech-ologies, and combines them in a new way in order to produce aovel point of care methodology for analysis of individual tear elec-rolytes. It is applicable to ex vivo lenses at the point of removal andlso to the in vivo tear meniscus. Since tear electrolytes exchangereely with the in vivo contact lens the freshly removed lens is

n ideal sampling device provided that it can be matched withn appropriate assay technique. It is applicable to any lens inde-endent of water content because the adherent tear film (or “tearnvelope”) is sampled [36].

coupled to a “U” bend PMMA fibre optic probe in contact with an ex vivo contact

The value of a tear sampling technique that simply involvesremoval of a lens has some clear advantages. It is quickly accom-plished and does not involve unwanted stimulation or reflextearing, with consequently enhanced flow rates which lead toerrors in the assay process. This problem is illustrated by measure-ments of total protein levels which are a useful marker of changestear composition and have been shown to be significantly higher intear samples collected at low flow rates compared to higher flowrates because of the dilution effects of reflex tearing [37]. The sameeffect has been demonstrated with serum albumin levels whichwere found to decrease from 750 �g/ml at an undisturbed flowrate of around 1 �l/min to 250 �g/ml at a stimulated flow rate of20 �l/min [38].

In this paper we describe a technique that combines develop-ments in fibre optic sensing technology with the use of a freshlyremoved contact lens and has the potential to be used in routineoptometric practice to give immediate point of care results. Thedevelopment of a potassium sensor is described here, but this isa platform technique applicable to a wide range of water-solubletear components.

2. Materials and methods

2.1. Total sensor system

A photograph of the sensor system is shown in Fig. 1 and aschematic layout of the component parts in Fig. 2.

Fig. 2. Schematic layout of the sensor components shown in Fig. 1.

1 & Anterior Eye 35 (2012) 137– 144

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40 D. Harvey et al. / Contact Lens

hemically specific detecting agent is placed in contact with thenalyte (e.g. lens or tear sample). If the analyte causes a changen the sensor coating (colour or precipitate for example) a pro-ortional change in signal is detected. The signal processing andlectronic control is handled by a software package installed on aonventional laptop computer linked to the sensor box.

There are three separate but related elements of the sensor sys-em:

the fibre optic and probe configurationthe sensor coating and sensing agentthe electronics – sensor box and computer software.

.2. The fibre optic and probe

Although optical fibres have been available for many years, earlyaterials had shortcomings that limited their application in dispos-

ble routine point of care situations. Early polymer optical fibresere of insufficient quality to be used in a sensing application, due

o their poor attenuation characteristics. Consequently glass opti-al fibres were preferred, and lasers producing as they do, moreoherent radiation, were the preferred light source for fibre opticork. As a result, for many years, the cost and size of laser light

ources and associated detector technology, made disposable pointf care fibre optic sensing impractical. However, as technology hasrogressed, the quality of coherent light from cost effective lightources has improved, as have the detectors. The size of a com-ined LED light source and detector all-in-one unit can be as smalls a mobile phone. This can be connected to a conventional laptophich operates control software which additionally processes theata. These factors have facilitated the use of the high quality yet

nexpensive polymer optical fibres which are now available for theevelopment of cost-effective sensor systems.

The key property exploited in fibre optic sensor technology isotal internal reflection, which is an optical phenomenon observedhen light is launched down the fibre. For total internal reflection

o occur, the following criteria must be met. Firstly, the refractivendex of the medium outside the fibre core should be lower thanhat of the core of the fibre, which means that the light will not passut of the core of the fibre, but will be reflected back into the fibre.econdly, the angle of incidence at which the light hits the boundaryf the fibre should be less than that of the critical angle of incidence39]. The critical angle is governed by the relative refractive indicesn the core and external medium. Any change in the refractive indexf the external medium will result in a change in the loss processt the interface.

Polymer-based optical fibre was traditionally fabricated fromoly(methylmethacrylate) (PMMA) core material, clad with fluo-inated polymers. Higher performance copolymer fibres are nowvailable but the reflection principles apply equally. Fig. 3 illus-rates the core and cladding of the fibre, which is flexible andan be formed into coils and bends as shown. When sections ofhe cladding layer are removed the clear core is exposed allowingccess to the evanescent field of light launched down the fibre. Thexposed area forms the sensing region.

The simplest form of sensor is achieved by bending and warm-ng the fibre into the form shown in Fig. 3. A section of the claddingayer is then removed in the region of the bend with a suitableolvent, taking care not to damage the core. This is convenientlychieved with an acetone-soaked tissue. Fig. 3 illustrates this pro-ess. The exposed area can then be coated with a suitable sensing

gent which on contact with the target analyte will change, forxample, in colour or refractive index. Such changes will produce aignal modulation through alteration of the intensity of the incidenteam.

“U” bend. The central PMMA core of the fibre is blue and the fluorinated polymercladding layer is black. On the right is a schematic representation of a PMMA fibrewith cladding layer removed to expose the sensing region of the fibre core.

2.3. The sensor coating and sensing agent

Removal of the cladding and replacing this by a coating that isresponsive to the external environment offers the opportunity tomodulate the loss of light at the interface by the response of thecoating. There are three ways in which that may be achieved in thesystem described here. They are:

• a change in the refractive index of the coating• a change in colour of the coating• a change in reflectance or light scattering ability of the coating.

In this assay we need to monitor the change in one componentof the environment – the target analyte, in this case the potassiumion. For that reason the sensor coating must be freely permeable topotassium ions. Additionally it needs to contain within its structurea component with which potassium ions will react in order to pro-duce a change in one of the three aspects of behaviour describedabove.

A logical choice of coating material for these applications is ahydrogel polymer. Hydrogels are water-swollen networks capableof exhibiting a wide range of properties, particularly permeability,depending upon the monomers used for their synthesis. They canbe synthesised as linear soluble polymers, coated on a substrateand cross-linked in situ.

The equilibrium water content (EWC) of the hydrogel will deter-mine whether or not specific tear components can penetrate.For sodium and potassium an EWC of at least 20% and prefer-ably somewhat higher is desirable. Rather than chemically bindingthe sensing molecule physical immobilisation is preferred whichentails selection of appropriate monomers units and cross-linkingsites to achieve the necessary degree of hydrophobic locking. Thedetails of sensing agent, backbone monomers and cross-linkingsystem used in the fabrication of a potassium sensor coating aredescribed in Section 3.

2.4. The electronics – sensor box and computer software

The components of the sensing system are shown in Fig. 1. Thelight source and detector are housed in a single box – the propri-etary EvanesCo EV 5000. An optical fibre cable connects the lightsource and detector, passing through the sensing region, whichmodulates the signal. The LED light source has three channels,red, green and blue which can be selected interchangeably. Fig. 1shows the software display on the laptop screen, from which thebox is controlled. The signal is shown on an analogue dial and also

displayed graphically.

The output is set to zero and the sensor probe placed in contactwith the analyte which generates a signal difference in a sequenceof steps. The first step is the contact between (e.g.) an ex vivo lens

D. Harvey et al. / Contact Lens & Anterior Eye 35 (2012) 137– 144 141

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ig. 4. Displacement of sodium from water-soluble sodium tetra phenyl borate byotassium to form insoluble potassium tetraphenylborate.

nd the sensor coating. This allows the target tear analyte (e.g.otassium) to enter the coating and contact the sensing moleculehich causes a colour change, a precipitate with the sensor coating

r a change in refractive index. This change at the surface of thebre typically causes light to be lost and a consequent differenceetween the light emitted from the light source, and the light thateaches the detector in the sensor unit. This information is handledy the data processing unit, resulting in a signal loss measurementisplayed in decibels (dB). For a dedicated (e.g. potassium) sensorhe display would give a direct concentration measurement.

. Results

.1. Development of a potassium sensor

In order to adapt the principles of the system described in Sec-ion 2 to a functioning potassium sensor it was necessary first todentify a reagent or chemical species that would react specifi-ally with potassium ions to produce a tangible change that isapable of influencing loss of light at the sensor interface. A sec-nd requirement was a means of immobilising that reagent athe sensor/analyte interface. This required an optically clear coat-ng through which potassium ions can diffuse but from which theeagent would not escape.

Traditional “wet” analytical chemistry has identified specificolourimetric reactions (sometimes called spot tests) that enablendividual metals to be identified and quantified. For potassiumnd the related metals such as sodium and lithium no such defini-ive colourimetric reactions exist. Fortunately it is possible to useheir size and charge characteristics to form specific complexesith specific complex anions or chelating compounds.

The reaction that we have used for quantification of potas-ium in the presence of sodium is illustrated in Fig. 4. It dependspon potassium displacing sodium from a water soluble sodiumomplex with the tetraphenylborate anion to form a precipitatef the water insoluble potassium complex. Since potassium has aower electronegativity (0.82 Pauling units) compared to sodium0.93 Pauling units) the potassium ion readily displaces the sodiumon. When the resultant white precipitate is dispersed in a water-wollen hydrogel film it scatters light, consequently altering theight loss from the sensing region of the fibre optic and producing

change in signal proportional to the amount of precipitate.The structure of the tetraphenylborate anion with four phenyl

roups attached to a central atom makes it relatively easy to immo-ilise by hydrophobic locking. This is achieved by designing aydrogel with two key characteristics. The first is a water con-ent above the minimum required in order to allow free diffusionf sodium and potassium ions; a value of around 30% is morehan adequate to achieve this [40]. The second is the incorpora-ion of hydrophobic segments, preferably pendant phenyl or benzylroups, in the polymer backbone.

To achieve these objectives a linear polymer was synthe-ised from N-benzyl, N-methyl acrylamide; methyl methacrylate;-vinyl, N-methyl acetamide and hydroxethyl acrylamide. Theolymer was formed by free radical solution polymerisation using

Fig. 5. Signal loss vs. time on placing the sensor probe into a 200 mg/100 ml potas-sium chloride solution.

AIBN as the initiator at 60 ◦C, in a nitrogen atmosphere to preventinhibition of the reaction. To add cross-linking sites, the linear poly-mer is functionalised with N-methylol acrylamide which is addedat a concentration equivalent to the hydroxyethyl acrylamide. Theresultant solution is heated for two hours at 60 ◦C in the presenceof a trace of hydrochloric acid catalyst. The product solution isthen ready for the addition of the sensing molecule, a cross-linkingagent (typically polyethylene glycol diacrylate) and photoinitiator(Irgacure 184). The resultant solution is applied to the sensor probeand exposed to UV light for five minutes producing a hard sensorfilm ready for use. The coated sensor probe may be stored dry, orhydrated in water or potassium-free saline.

In order to test the efficacy of the sensing coating, the “U”bend configuration illustrated in Fig. 3 was used. A series of potas-sium chloride solutions was prepared and the response of fibreoptic sensor system was monitored. Fig. 5 illustrates the speedand stability of response when the probe is placed into the testpotassium chloride solution. The assay data is collected continu-ously and the output presented continuously on the laptop display(Fig. 1).

The range of calibration solutions was chosen to cover the rangeof normal potassium levels reported in tears and extend to therange of values encountered in urinary excretion (25–120 mEq/l).The signal loss plotted against potassium chloride concentra-tion (Fig. 6) indicate that in terms of range and sensitivity thetertraphenylborate-based fibre optic sensor offers the potential tofunction in point of care use for clinical samples. Blank experimentswith non potassium-containing electrolyte solutions (e.g. NaCl) ofequivalent concentrations indicate that this is a potassium-specificresponse and not a electrolyte-refractive index effect.

3.2. Probe configurations

An important variable in this type of sensor system is the probeconfiguration. For many applications a simple looped or “U” bendprobe is satisfactory, sensitive and very inexpensive to produce.This type of probe is useful for immersion in liquid samples but,equally, it provides a very convenient method for measuring tearpotassium levels associated with an ex vivo contact lens (Fig. 6).The in-eye lens equilibrates with tear electrolytes and on removalthe associated “tear envelope” provides a very useful substrate forpotassium assay.

An alternative type of configuration to the looped sensor, inwhich light from the source passes along the fibre and returns to thedetector, involves launching light down a single probe with a reflec-tive end which reflects and returns the light from the probe tip. This

type of probe, which is illustrated in Fig. 7A, is almost half the diam-eter of a contact lens and offers an alternative way of interrogatingan ex vivo lens.

142 D. Harvey et al. / Contact Lens & Anterior Eye 35 (2012) 137– 144

Fig. 6. Relationship between signal loss and potassium chloride concentration, and a photo of a contact lens in contact with a looped sensor probe.

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A further alternative, the single fibre probe format, involvesaking a reflective probe tip by cleaving the fibre shown in Fig. 3

nd polishing the tip. This configuration – a straight fibre probe –an be used to make direct measurements in the lower tear menis-us as shown in Fig. 7B. This is analogous to the technique usedith microcapillary tear sampling but does not involve removal of

ear.

. Discussion

This preliminary study illustrates the potential value of fibreptic sensing in point of care tear analysis. Such an approach hasany potential advantages. Tear analysis has been acknowledged

s a source of valuable diagnostic information for many years buthe problems of tear collection and storage are considerable whilsthe low volume available imposes severe limitations on the avail-bility of analytical techniques. A logical approach to this problem

re optic probe used for direct in vivo assay of tear in the lower meniscus.

lies in the use of contact lens wear as a sampling technique byremoval of the lens and the associated envelope of tear film [36].

Glucose is an example of a soluble tear-borne analyte whichequilibrates with the lens and generates real and ongoing interestas a target for a lens based assay [41]. Because of their size andsolubility tear electrolytes are equally accessible analytes in the exvivo lens and exploitation of this fact has been limited by lack ofa suitable assay method rather than failure to recognise the prin-ciple. Although use of the lens is the preferred method of samplecollection for the system described here, with an appropriately con-figured probe direct tear concentration measurements can be madevia the lower tear meniscus.

The case for the choice of potassium as an initial target tear

analyte has been made at the outset. Abnormalities in potassiumlevels have been linked to several clinical conditions, amongst themdry eye. An additional and important point is that the absenceof a convenient tear-based assay reduces the opportunity for

D. Harvey et al. / Contact Lens & Anterior Eye 35 (2012) 137– 144 143

Table 2Concentrations published for some of the major tear components in tears mg/100 ml [42].

Component Average concentration Max quoted value Min quoted value Number of reference sources

Sodium 338 354 327 7Potassium 82.7 137 58.7 10

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ecognising links between abnormal potassium levels and otherlinical conditions.

Examination of the number and range of literature values forear borne cations (Table 2) raises interesting points. It showsreater consistency (less variation) in the levels of sodium reportedhich may imply that techniques for the measurement of sodium

re themselves consistent and accurate. Alternatively it maybe,hat there is more genuine variation in potassium and calciumevels. In either case the availability of a convenient and inexpen-ive point of care assay technique for potassium (and subsequentlyalcium) can only improve the availability of data and clarify itssefulness.

The data recorded in Table 2 relate to concentrations of theotassium cation in mg/100 ml whereas the calibration graphhown in Fig. 6 is based on potassium chloride concentrationn mg/100 ml. Since the atomic weights of potassium and chlo-ine are similar (39 vs. 35.5) the potassium values in Table 1 arepproximately half those of the equivalent potassium chloridealue (i.e. 100 mg/100 ml potassium is equivalent to approximately00 mg/100 ml KCl).

The recent commercial availability of a point of care devicenabling measurement of tear osmolarity is proving increasinglyaluable in dry eye studies. The subsequent (September 2010) Tearilm & Ocular Surface Conference provided ample evidence thathe ability to monitor even such a basic property as tear osmolar-ty adds markedly to diagnostic capability and clinical debate [43].lthough tools to measure osmolarity are useful in providing anverall picture of soluble tear-borne components they do not pro-ide the detailed information necessary to link abnormalities inetabolic processes, to point of care clinical diagnosis.It is logical to expand the application of this platform technol-

gy to other tear analytes. A calcium sensor, possibly linked withagnesium, for example and would exploit exactly the same prin-

iples described here. In addition to its clinical value such a sensorould have practical utility in contact lens practice in contact lensaterial selection. The underlying causative factors leading to cal-

ium and white spot (“jelly bump”) formation has been knownor several years [35,44,45]. Elevated tear calcium levels form oneart of the equation and become problematic when linked withaterial/patient combinations that produce high levels of degraded

ipid.In summary several strands of technology have now come

ogether to enable inexpensive point of care fibre optic sensors toe produced. The versatility of probe configuration design, coupledith the ability to design a range of sensor coatings able to take

dvantage of the combined optics and electronics platform meanhat key tear analytes can now be assayed in the clinic, rather thanhe laboratory. There are inevitably questions of design optimisa-ion and product reproducibility that would need to be addressedor a mass-produced system to become a reality but the principlesre clearly illustrated in the prototype system.

A key feature of this emerging technology is that it would enablehe ex vivo lens to become a preferred method of both sample

ollection and presentation for a series of tear film assays. All thelements of this novel system are established in other applications,ut have not previously been brought together in a point of careiagnostic tool.

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