Ultraslow microdialysis andmicrofiltration for in-line, on-line andoff-line monitoringJakob Korf, Kirsten D. Huininka and Geertruida A. Posthuma-Trumpieb

University of Groningen, University Medical Center Groningen (UMCG), University Center Psychiatry (UCP) Section Biological

Psychiatry. P.O. Box 30.001 9700 RB Groningen, The Netherlands

Review

Glossary

Arterial venous (a–v) difference: difference of the analyte concentration of the

outflow vein and the incoming artery of an organ measured simultaneously.

Multiplication of the a–v difference with the blood flow gives the net rate of

uptake or consumption (negative values) or the production or release (positive

values) of the analyte of the organ (e.g. brain).

Biofouling: undesirable accumulation of cells or microorganisms on a wet

surface.

Biosensor: according to the International Union of Pure and Applied Chemistry

(IUPAC) a biosensor is: ‘‘a self-contained integrated device, which is capable of

providing specific quantitative or semi-quantitative analytical information

using a biological recognition element (biochemical receptor) which is retained

in direct spatial contact with a transduction element’’ [75].

Hydrophilicity/hydrophobicity: physical property of a molecule to attract/repel

water; often hydrophilic molecules are easily dissolved in water and

hydrophobic molecules in fat; they are lipophilic.

Lab-on-a-chip: stand-alone, fully integrated system to analyze samples without

user interference. A connection to a PC is usually provided to store the results

in an appropriate format, for example, a laboratory information management

system.

Microreactor: reaction vessel or configuration with volumes below 1 ml. It can

often be used as biosensor when placed in-line with a detector system (e.g.

electrochemical detector).

In medicine and biotechnology, close monitoring ofmolecular processes might assist to optimise thera-peutic interventions and production of biochemicals,respectively. Here, we summarize the current status oftwo automatic and continuous sampling technologies,microdialysis and microfiltration, which facilitate both invivo and in vitro monitoring of nearly any analyte,because they can be combined easily with many ana-lytical techniques. Conventional microdialysis andmicrofiltration, which require collecting relatively largesamples, are however often impractical and semi-quan-titative; hence, we focus on ultraslow sampling to cir-cumvent such limitations. Ultraslow microdialysis andmicrofiltration already have been used successfully forquantitative pharmacokinetics, glucose metabolism(e.g. of the brain), cytokines and proteomics (e.g. tumoursecretomes), both in vivo and in vitro.

The potential of in vivo and in vitro monitoringIn biological and medical sciences as well as in bioproces-sing, close monitoring of biomarkers or reactor constitu-ents, respectively, might help to optimize therapeuticinterventions and the production of biochemicals. Forexample, in biomedical sciences, exact knowledge of cer-tain indices of metabolism (e.g. glucose, lactate), hormones(e.g. cortisol) or cell growth (e.g. peptides and proteins)might help to optimize sport training and diabetes man-agement, or to assess the proliferation of cancer. In bio-technology, maintenance of constant levels of substrates,or a balanced withdrawal of reaction products, will maxi-mize yield. Close monitoring requires frequent and sterilesampling, often done with manual or semiautomatic pro-cedures. We consider ultraslow (flow rate < 0.5 ml/min)microdialysis (MD) and microfiltration (MF) as a valuableoption for automatic and continuous sampling of fluids.MDand MF (Box 1) are versatile and quantitative technol-ogies, and furthermore, the collected samples can be ana-lysed with a wide variety of analytical techniques [1–9].

MD was first introduced and commercialised in the1970s and was applied initially to analysis in the brain,although peripheral applications were soon recognized. To

Corresponding author: Posthuma-Trumpie, G.A. (truus.posthuma@wur.nl)a Present address: Brainlink B.V., P.O Box 4030, 9701 EA Groningen, The

Netherlands..b Present address: Wageningen UR, Agrotechnology and Food Sciences Group, PO

Box 17, 6700 AA Wageningen, The Netherlands..

150 0167-7799/$ – see front matter � 2009 Elsevier Lt

date, a large body of evidence is available that demon-strates the usefulness of MD and ultraslowMD, in particu-lar in clinical applications. To illustrate the wideacceptance of MD monitoring, a recent consensus grouphas discussed in depth the potential and limitations of MDfor pharmaceutical applications [1]. As far as we are aware,there are no reports on MD for industrial applications.

MF was first introduced as a sampling technology in1987 [10] and has been subsequently commercialized. Theterm ultrafiltration (UF) also has been used to denoteMF.It has initially particularly been used for in vivo monitor-ing of energy metabolism, drugs and electrolytes[3,5,11,12].

Quantification in MF and MD not only poses an ana-lytical problem (that is not discussed here in detail), butalso relies on the fact that the probes used (Box 1) consist-ently and reliably represent the analytes in vivo and invitro. In conventional MD applications, complicated pro-cedures are required to derive the tissue concentration ofan analyte from its concentration in the dialysate [13–21].Box 2 describes models used to calibrate the concentrationof the analyte in the probed compartment in vivo. At very

Microdialysis (MD): perfusion technique to recover analytes by diffusion over

a semi-permeable membrane.

Microfiltration (MF): withdrawal of a biomatrix (body fluid or reaction

medium) through semi-permeable membranes.

Ultrafiltration: older but frequently used term for MF.

Ultraslow MD or MF: MD or MF at rates below 0.5 ml/min.

d. All rights reserved. doi:10.1016/j.tibtech.2009.12.005 Available online 14 January 2010

Box 1. MD versus MF

MD is based on the diffusion of analytes over a semi-permeable

membrane of a continuously flushed implanted hollow fibre probe

(left panel, Figure I). The shape of the MD probe can either be linear

(single hollow fibre) or concentric (the outer tube is semi-permeable

fibre and an inner tube provides the perfusion fluid). Probe length

varies from 0.1 to 10 cm and its volume is about 0.1 ml/cm length.

The perfusion fluid has to be pumped using mechanical pumps,

which makes the use of electrical power obligatory. In a typical set-

up, the outflow (the dialysate) contains the analytes of interest.

Fractions are collected manually or automatically (with a fraction

collector) at times between 1 and 30 min. Major advantages of MD

are the relative ease to set up the system, its versatility to a variety of

analytes and analytical tools, the possibility to maintain sterility

during sampling, worldwide experience with the technique, as well

as its commercial availability and professional support to new users.

At very low perfusion rates (< 0.5 ml/min) MQMD (middle panel,

Figure I) is a convenient alternative to MD. In MQMD, the outflow is

diluted before transport to an analytical device. Using MQMD, the

transport time of the analyte is reduced, however, more sensitive

analytical techniques are obligatory because of the resulting sample

dilution.

MF uses a generated pressure gradient over the MF probe

membrane, which allows removal of filtrate from either body fluid

(in vivo), or medium (in vitro) in discrete or continuous samples

(right panel, Figure I). Thus, the concentration of the analyte in the

filtrate approaches that in the probed biomatrix. Apart from its

ability to provide quantitative analysis, another advantage of MF is

that no energy-consuming (electromechanical) equipment is neces-

sary to maintain a constant influx (as with MD) for sample

withdrawal. Continuous MF sampling at low withdrawal rates

(<0.1 ml/min; ultraslow MF) can be also achieved without electro-

mechanical equipment. A limitation of MF in vivo might be the

production rate of the probed body fluid, which is particularly

apparent in the rodent brain. In the human brain, these limitations

are not encountered because larger probes are feasible and the

extracellular space is relatively large.

Figure I. Configurations of concentric MD, MQMD and MF probes.

Review Trends in Biotechnology Vol.28 No.3

low perfusion rates, the levels of the analyte in the sampleand in the external milieu are (nearly) identical. Slowperfusion, however, is considered unattractive becauseonly a very small sample volume and thus small amountof the analyte can be collected, and moreover, sampletransport to the analytical or collection device is slow.Diluting the dialysate with a carrier fluid has circum-vented these limitations. By so doing, the transport ofthe sample from the probe to the detection device is shor-tened substantially (often up to eightfold). Thismethod hasbeen termed metaquant microdialysis (MQMD) [22,23],and is summarized in Table 1, together with the potentialof MD and MF.

In contrast to other overviews of MD and MF [1–9], thisarticle focuses on novel developments of sampling at ultra-slow flow rates. We discuss here in depth the potential andlimitations of ultraslow MD and MF for quantitativemonitoring. We highlight recent advantages in the devel-opment of new probematerials for collecting high- and low-molecular-weight compounds and also comment on anyissues associated with biocompatibility. We discuss how tocollect and store submicrolitre samples for online and off-line analysis. Finally, we summarize recent biomedicalapplications and the unexplored potential of ultraslowMD and MF. We evaluate ultraslow MD and MF technol-ogies in Table 2.

Probe materialsThe membrane in MD and MF probes is the boundarybetween the external and the internal compartments.Probe membranes should on one hand provide maximalrecovery of the analyte while minimizing the accumulationof constituents that could interfere with the analysis. Inprobe design, there are four major issues to consider: (i)their possible preference of hydrophilic versus hydrophobicanalytes; (ii) their molecular weight cut-off (MWCO); (iii)compatibility of the material with the compartment to beprobed (this issue is in particular important for biomedicalin vivo applications); and (iv) robustness and geometry ofthe probe allowing manual handling.

In most of the applications, probes are placed in ahydrophilic environment for the collection of low-molecu-lar-weight hydrophilic analytes. It is obvious that onlyanalytes with appropriate hydrophilicity and MWCOvalues for the respective membrane can be analysed. InMD, cuprophan (MWCO6 kDa) is themost frequently usedcommercial membrane, which exhibits an almost neutralsurface. For the recovery of charged analytes, polycarbo-nate–polyether, polyacrylonitrile and cuprophan mem-branes have given satisfactory results [20]. For classicMF, the successful use of different membrane materialsincluding polysulfone (MWCO 100 kDa), polycarbonate–

polyether (MWCO 20 kDa) polyacrylonitrile, acrylonitril–sodium methallylsulfonate, or polyethylene coated withethylenevinylalcohol polysulfone (range MWCO 15–

50 kDa) has been reported [24,25]. However, systematicinvestigations on the usefulness of various probe mem-branes for ultraslowMDandMQMDaremainlymissing [8].

For ultraslow MF studies, in which it was important toimprove blood compatibility, other materials have beenused, including polyethylene coated with ethylenevinylal-cohol (‘‘plasmaflo’’), polyethersulfone (‘‘microPES’’ and‘‘transvivo’’) and polysulfone (‘‘plasmacure’’) coated withheparin to prevent blood clotting. The efficacy of theseprobes for the collection of hydrophobic hormones (cortisoland corticosteron)hasbeenassessedboth in vivoand in vitro[26,27], and only in vitro for various amino acids and hydro-phobic and hydrophilic peptides and proteins [27]. Poly-ethersulfone (MWCO 30 kDa) and regenerated cellulose(18 kDa) havebeen tested in vitro inMQMDfor themonitor-ing of brain-targeted drugs [22]. The best results have beenobtained with polyethylene coated with ethylenevinylalco-hol (‘‘PE-EVAL’’) in MQMD experiments, in which almost100% recovery of citalopram and cortisol was achieved in

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Box 2. Equilibrium MD and modelling

Essential for the interpretation of MD results and the optimization of

the method is the efficiency with which the analyte passes through

the probe membrane, that is, its mass transfer (often noted as

recovery) [13–21,72–74]. Mass transfer over the probe membrane

depends on the geometry and the properties of the membrane as well

as the perfusion rate. Models are necessary to calculate (or estimate)

the concentration of an analyte in a tissue or in the medium as

exemplified by the following two case studies.

Case study 1

Diffusion of low-molecular-weight hydrophilic analytes is relatively

unrestricted in body fluids or in vitro. The probe is a concentric

configuration of hollow fibres with an exposed surface that allows the

passage of the analytes and an inside inflowing tube (Figure Ia). It can

be assumed that the diffusion rates over the membrane are identical.

At zero flow rate, the concentration of the analyte (Ctin) at time t in the

perfusate is:

Ctin ¼ kCoutð1-e�kAtÞ;

where A is the area and k is the bidirectional diffusion constant of the

analyte in 1 (min mm2)�1 (Figure Ia, lower figure). Equilibrium is reached

when the concentration of the analyte outside and inside the probe is

identical (Cout = Cin).Consideringthevolumeofthenetinternalvolumeof

the probe as half of the inner volume, the average residence time of the

perfusate is tr = V/2F, where V is the volume and F the flow rate in ml/min.

Foraprobewithanexposedtipof5 mmandaninnerdiameterof100 mm,

the tr at a flow of 0.1 ml/min is approximately 10 s. Hence, in about 10 s,

near equilibrium is reached. In the case of high-molecular-weight ana-

lytes and probes with large and asymmetrical pores, the diffusion coeffi-

cient for inwarddiffusionmightbehigher thanthat foroutwarddiffusion;

when ki>>ko a nearly linear accumulation of the analyte occurs. Under

these conditions, collection of the extracellular matrix by MF with the

guaranteed near 100% recovery might be the best approach.

Case study 2

For in vivo tissue sampling, the situation is more complicated (Figure

Ib). Firstly, insertion of the probe creates an (artificial) extracellular

space, and secondly, the diffusion of the analyte in tissue is limiting or

conversely, the analytes originate in relatively large pools. Ultraslow

MD can overestimate the interstitial concentration [1,4]. As indicated

in Figure Ib, lower figure, the contribution of more remote tissue to

the composition of the dialysate decreases exponentially. In practice,

the cell layer around the probe will contribute most if not exclusively

to the analyte in the outflow, even at equilibrium dialysis [37].

Figure I. Diffusion characteristics of the analytes for in vitro and in vivo sampling.

Arrows indicate trajectories of the analytes through the biomatrix surrounding the

probe and over the probe membranes. (a) In vitro sampling using a concentric

configuration of hollow fibres with an exposed surface allowing the passage of the

analyte and an inside inflowing tube (top). In the graph below, residence time at

zero net flow rate is indicated. (b) Experimental set-up of in vivo sampling from

tissues (top). As shown below, the contribution of tissues, which are more

remotely located from the probe, to the composition of the dialysate decreases

exponentially.

Review Trends in Biotechnology Vol.28 No.3

vitro. The in vivo performance of all the membranes wasfound to improve substantially (i.e. recovery became near100%) by heparin coating, not only when used within theblood circulation, but also subcutaneously or intraperitone-ally (Huinink, K. D. 2009, PhD thesis, University of Gronin-gen: http://irs.ub.rug.nl/ppn/318767716).

Another factor to consider is the robustness of the probe,which depends, among other things, on the robustness ofthe membranes; hence, a minimum probe size of 0.1 cmoften is used. The small size of MD and MF probes allowsfor the monitoring of reactions in vessels with volumessmaller than 100 ml. Sterile sampling (to prevent contami-

Table 1. Comparison of MD, MQMD and MF

Parameters MD/ MQMD*

Driving force Mechanical pump

Method of analyte retrieval Diffusion

Retrieved samples Sterile, can be with or

proteins as desired

MWCO hollow fiber 0.1–3 kDa;

>>MW water leakage

In vivo sampling All tissues

Sampling rate (range, ml min�1) 0.5–1/0.05–0.5

Sampling frequency > min

Recovery/calculation after retrieval Often unknown/100%

Local delivery of compounds Yes

Used in clinic/human MD used both in hum

animals; MQMD only*Table is composed from earlier reviews [3–6,8] and literature presented here.

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nation) and monitoring is particularly useful in the optim-ization of the production of compounds that are rare,expensive or novel within provisional settings on thelaboratory scale. Although the semi-permeable membraneof a probe is rather fragile, some measures can be taken toincrease robustness. For example, insertion of a metalspring inside the probe not only reduces the volume ofthe probe, but also decreases its fragility [3,28]. Placementof the probe inside a preferentially flexible hollow needlemight also increase its robustness, as in this set-up, onlythe tip of the probe membrane is exposed (similarly toclassical MD) [7].

MF*

Under-pressure/vacuum

Filtration

without Sterile, can be with or without

proteins as desired

0.1–100 kDa

from probe

Most tissues (not rodent brain)

0.05–1

Continuous

100% recovery

No

ans and

used in animal studies

Occasionally

Table 2. SWOT analysis of ultraslow MD, MQMD and MF

Strengths Weaknesses

In contrast to conventional MD, both ultraslow (equilibrium) MD and

MF give the actual concentrations of the analyte as in the tissue,

biomatrix or medium; hence, complicated calculations are avoided.

Main challenge is the handling of small samples (often <100 nl) that

contain only small amounts of the analyte; hence, for most applications,

high detection sensitivity is required.

Because of the equilibrium there is no concentration gradient of the

analyte over the probe membrane. Hence the concentration of the

analyte in the MF or MD is (without pre-concentration) the highest

possible.

There are no commercial suppliers and support for dedicated

ultraslow sampling (except for MQMD).

MQMD combines advantages of ultraslow sampling (measuring

the actual concentrations in the probed compartment) with that of

conventional MD (avoiding small sample handling to an analytical

device).

The MQMD sample is diluted approximately 5- to 10-fold to

concentrations that can become limiting in some analytical

devices.

Methods for in-line/on-line/off-line sample analysis have been

described, both as proof-of-principle and in some cases in real

biomedical applications.

Ultraslow sampling is unsuitable for sub-minute monitoring,

or for environmental monitoring, when low amounts of analytes

are found in large volumes.

Both conventional and ultraslow MD and MF allow continuous

sterile sampling. For ultraslow MD and MF, devices for integrated

collection and storage have been described.

Although numerous applications of MD/MF have been published,

most of them only presented proof-of-principles with only few

demonstrated real applications

Low-energy, non-mechanical pumping is feasible with ultraslow MD

and MF, but not for conventional MD. Low-energy devices provide

pulsation-free flow and can be produced from inexpensive materials.

MD and MF are invasive techniques. Hence there is risk of infection

of the subject (human or animal) during long-term applications in vivo.

Ultraslow sampling techniques can be used in wearable monitoring

devices in natural environments.

Suitable for processes with time resolution ranging from minutes

up to hours.

As with conventional MD and MF, depending on the analytical

technique used, in principle, many if not all types of analytes can be

monitored. In vivo and in vitro applications have been described for

proteins, peptides, hormones, electrolyte ions, energy metabolites,

amino acids and many drugs, either free or protein-bound.

Specific applications are possible after relatively easy adaptations

of commercial conventional MD/MF probes.

Commercial options and support available for conventional MD,

MF and MQMD.

Opportunities Threats

Many of the disadvantages are opportunities for development of

new equipment. The most innovative developments include

integration with mass-produced microchip technology.

Better performance and biocompatibility of implantable

(needle-shaped) biosensors in dedicated applications. Such sensors

could replace MD and MF in some applications. Relevant for the

management of diabetes (glucose) and in the intensive care

(e.g. glucose, lactate and oxygen).Improvement and widening the versatility of pumping systems.

Automatic biomedical sampling (e.g. blood in the intensive care).Possible introduction of electro-osmotic, osmotic, micro-electrical

(mini)pumps and adaptation of medical drug delivery pumps.

Use of analytical equipment dedicated for nl samples, including CE,

MS, and microbore LC; possibly integrated with microchip

technology.

Non-invasive detection methods (e.g. glucose with infrared, or

oxygenation of blood).

Thus far, there have been few scientific, technical and commercial

efforts in developing biomedical applications in vivo and process

chemistry applications in vitro.

Many patents for conventional MD have expired and there are only

few patents dedicated to ultraslow MD and MF.

Review Trends in Biotechnology Vol.28 No.3

In in vivo applications, probe implantation can causewound reactions, either because of clotting and subsequentadherence of proteins and cells to the probe (in blood), or toan inflammatory response in tissue [29–31]. Biofouling ofthe probe is, apart from the material used, also dependenton the size of the membrane pores [32–38]. Blood (andother tissue) compatibility of a probe can be substantiallyimproved by covering its membranes with a layer ofheparin [36].

These studies emphasize that the suitability of a probemembrane depends both on the analyte and the appli-

cation. In addition to more obvious parameters, such asMCWO and hydrophilicity, heparin coating also shouldbe considered for in vivo applications that do not involveblood.

Pumping, handling and storage of small samplevolumesUltraslow equilibrium MD and MF require pumping atvery low speed. Commercial mechanical MD (syringe)pumps can achieve rates lower than 1 ml/min, but at ratesbelow 100 nl/min, the flow might become irregular. Very

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Figure 1. Typical configurations of probes used for MQMD, and for MF storage capillaries. On the left, a complete MQMD probe is shown and important details of the flow

confluences and the membrane are shown magnified [49]. The flow rates of dialysis and carrier fluids and their directions are as indicated. Reproduced with permission

from Ref. [22]. On the right, the actual realization of an ultrafiltration probe coupled to a storage capillary and a syringe (the vacuum pump) is shown. The vacuum pump is

in fact a disposable syringe, in which the resistance of a restriction capillary placed between the storage capillary and the pump determines the actual flow rate.

Review Trends in Biotechnology Vol.28 No.3

low and pulsation free perfusion flows easily can beachieved with non-mechanical devices. We have used pre-viously a pumping system that is based on partial vacuumcombined with a capillary fluid resistance, to control flowrate (Figure 1) [3,5,39,40]. Such devices are inexpensivebecause they can be constructed from medical disposables.As a result of their low energy use, these devices easily canbe adapted for applications in freely moving subjects, asillustrated recently in chickens and cows [26,40]. Otherpossibilities, although not yet explored at present, areimplantable mini-pumps based on osmotic gradients,which are also used for slow delivery of drugs [41,42],and electro-osmotic pumps that are based on the drag forceexerted on an electrolyte by double layer chargesmoving inan electric field [43]. The latter pumpmight be switched onand off by remote control. The requirement of an externalpower source might, however, limit the development of awearable format, although promising developments in thisdirection have been reported recently [42,43]. The adap-tation of externally controlled medical devices, such asthose used in patients for the slow infusion of therapeuticdrugs against cancer or spasticity, could also be considered.

In conventional MD, the dialysates are collected eithermanually or in small-sized fraction collectors. However,using ultraslow collection techniques, the resulting frac-tions are often too small to be handled manually. Thus, theprobe outflow might be coupled directly to an analytical

154

device, such as liquid chromatography (LC). However, evenhere, relatively large samples (>1 ml) are often necessary,because of the large dead volumes caused by the connec-tions of the various parts of the equipment and con-sequently long delays. Therefore, in some cases,miniaturized biosensors have been positioned in-lineimmediately after the probe to enable in-line analysis[44,45]. Analysis delays are circumvented with MQMD[22,23], in which the probe outflow is diluted with a carrierfluid by a fixed ratio (e.g. 10-fold) (Figure 1).

Another approach to handle nanolitre samples is tostore MD or MF samples in a capillary tube for subsequentanalysis. The outflow of the MD or MF probe is pusheddirectly into the storage capillary, hence, any temporal invivo variation is maintained during storage. To avoidliquid swivels (with relatively large dead volumes) in smallfreely moving animals (rodents), the capillary can hangfree above the animal cage. A sample of 100 nl occupiesabout 1 cm in a capillary of 100 mm diameter, and capil-laries of 3–6 m length have been used successfully forsample storage [39,40]. Diffusion in the transversal direc-tion is negligible and has been shown to be <1 cm/day forsmall hydrophilic analytes, and is substantially less forproteins because of their diffusion constants in water [27].Moreover, as a result of the charge of the glass capillary,which is the most often used material, the diffusion ofwater, and accordingly that, of solutesmight be even lower.

Review Trends in Biotechnology Vol.28 No.3

For example, samples that contain glucose, lactate orcortisol diffused in the capillary to an extent that wasequivalent to less than a 5-min collection time after 24 hstorage at 4 8C. The formation of bubbles in the capillaryduring storage over longer periods can frequently be aproblem, but has been found to be negligible when fusedsilica tubing (FST) is used [40]. In comparison, polyethy-lene tubing with an outer wall of PVC shows many emptyspaces, which presumably are air bubbles. Evaporationand air bubbles can be avoided in more porous tubingmaterial (e.g. pure polyethylene) only when the tube iswrapped additionally in a moist plastic bag [39]. We thusconclude that FST is the best material for storing samples[27,39,40]. Such fused silica capillaries also were the pre-ferred capillaries in proteomics studies by Huang and co-workers [46–48]. As a result of the virtual absence ofartefacts arising from diffusion effects, capillary-storedsamples can also be sent to other laboratories for analysis,which presents another significant advantage. Foranalysis, the sample can be simply pushed out of thecapillary into an analytical device.

Taken together, these studies clearly point towards thepotential of non-mechanical pumping systems combinedwith the storage of samples in fused silica capillaries thatallow later chemical analysis as a versatile modality forhandling sub-microlitre samples.

ValidationThe gold standard of validating MD and MF is by compar-ing the analyte concentration in the probed extracellularcompartment as obtained from standard techniques withthat measured in the microdialysate or microfiltrate. Suchvalidations usually are possible in vitro, but are onlyfeasible in vivo when the probes are placed in body fluidcompartments. In medical practice, blood, as well as cere-brospinal, lymph, saliva and synovial fluids can be used fordirectly comparing the concentrations of the analyte in thedialysate or filtrate with those obtained by standard tech-niques.

When such standardmeasurements cannot be obtained,other validation methods are necessary (Box 2). For con-ventional (high rate) MD, these include: (i) equilibriumMD, (ii) the no-flux method [17], or (iii) retrograde dialysis[13]. The no-flux method is based on the principle that,when the concentration in the dialysate (Cin) is the same asthat of the perfusate (Cout), there is no net transport of theanalyte over the probe membrane, and accordingly, theconcentration in the tissue compartment is identical(Cin = Cout) [17]. In a retrograde dialysis set-up, the out-ward diffusion of an internal standard, which is verysimilar to the analyte, is measured and it is assumed thatthe resulting rate reflects inward diffusion of the analytefrom the intercellular space. In this method, the concen-tration of the analyte in the dialysate is multiplied by theratio of the concentrations in the in-flowing and out-flowinginternal standard and this amounts to the presumed con-centration in the body compartment [13]. One could alsocompare ultraslow MD results with those obtained withthe retrograde or the no-flux methods. The no-flux methodhas been used successfully to validate MQMD for pharma-cokinetic studies [22,23,49]. It should be emphasized that

validation methods used for conventional MD are timeconsuming and therefore preferentially applicable understeady state conditions [13–21]. A more simple way tovalidate ultraslow MD is by using two or more perfusionspeeds in the sub-microlitre-per-minute range, and at equi-librium, Cout should be independent of perfusion rate.Another approach for validation is to compare analyteconcentrations obtained by ultraslow MD with thosemeasured by MF. For example, a rat study has shownidentical subcutaneous levels of glucose and lactate in theoutflow after switching fromMD toMF, and vice versa [50].

A problem with in vivomonitoring is that the size of theextracellular compartment can change during the obser-vation period, for example, during hypoxia of the brain[51]. The cellular and consequently the intercellularvolume might also change during non-pathological phys-iological activity [52].

As outlined above, quantitative monitoring with MDrequires validation methods, that is, comparison withaccepted approaches. Most of these approaches requirecomplicated procedures and are in practice only applicableunder steady state conditions (Box 2). A relatively simplemethod to validate ultraslow MD that is also applicable tonon-steady state conditions is to show that the concen-tration of the analyte in the sample is independent of the(low) flow rate.

Analytical techniquesIn conventional MD, microlitre samples are subjected todiscrete analysis based on liquid chromatography andoptical (fluorescence, UV) or electrochemical detection.Alternative analytical methods use biosensors and micro-reactors, or more recently, capillary electrophoresis (CE)and mass spectrometry (MS). The main challenge associ-ated with these methods is how they can be adapted to beapplicable for ultraslow MD and MF.

A few reports have discussed the in-line application ofbiosensors. Considering the low flow rate and the smallvolumes, biosensor detection must be possible in nanolitrevolumes. For example, successful in vivo application ofbiosensors with sample volumes<50 nl have been reportedfor glucose and lactate that had been collected with ultra-slow MF in the 0.5–20 mM range. In these studies, needle-shaped sensors were used that had been covered in situwith the oxidating enzymes used for detection (glucose orlactate oxidase respectively) and 1,3-phenylethylenedia-mine by electropolymerization [44,45]. Monitoring inmicrolitre volumes is possible with microelectrodes madefrom glass capillaries with a tip diameter�50 mm, but suchapplications have not been explored inMD andMF. Onlinedetection with sensor configurations that are based onmicroreactors is another option [53,54]. Here, using micro-valves, nanolitre samples from the MD or MF outflow areinjected into a buffered stream and led through a micro-reactor that contains an excess of enzyme to produceelectrochemical products. Such devices have been shownto give reproducible signals for months after the analysis ofmore than 10 000 samples [53,54].

The simultaneous analysis of several analytes can beachieved by combining ultraslowMDorMFwithmicroboreHPLC, MS, CE or with protein microarrays. In particular

155

Figure 2. Example of experimental MF set-up. The example shown here is that of a

capillary ultra-filtration for the continuous monitoring of proteins released from a

subcutaneous probe in the ear of a mouse. (a) This panel shows the MF probes (1)

attached to a fused silica capillary (2); polytetrafluoroethylene ultramicrobore tube

(3) connecting the ultrabore tube (4) and a sharpened 27-gauge needle (5) that

ends in a collection tube (vacutainer). The magnification of the semi-permeable

hollow fibre (probe) is shown in the inset; scale bar: 7.5 mm; (b) schematic

illustration of the probe set-up. (c) This panel shows the actual experimental set-up

with a schematic representation (d). A vacutainer (6) (a tube with rubber stopper

used for routine collection of blood) provides the under-pressure (8); 7

counterbalances and fixes the vacutainer. Details are given in [48]; reproduced

with permission.

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integrated lab-on-a-chip devices are currently the mostchallenging options [55]. Recently, ultraslow collected dia-lysates have been subjected to subsequent tandem MSanalysis to study cortisol [56] or carbathione [57]. Micro-bore HPLC and CE can be used for multi-analyte analysisof drugs, amino acids and neurotransmitters in vivo[55,58]. In cases for which no optical or electrochemicaldetection is possible, either pre-column or post-columnderivatization with a chromophore or electrophore isnecessary for detection [55]. CE is fast (10–120 s) andrequires only picolitre volumes of microdialysate or micro-filtrate. A micro-fabricated fluidic electrophoresis device(microchipE) coupled to online MD for primary amines isbased on electrochemical detection [59] or pre-columnderivatization with o-phthaldialdehyde (for laser fluor-escence detection), a flow gated injector and a separationchannel [58,60]. Smearing of the (aqueous) MD sampleswas suppressed by introducing segments of organic per-fluordecalin in a microfluid chip [61]. The segmented flowset-up was combined with CE for amino acids [61].

Most methods described above are in the stage of proof-of-principle and only few proven applications have beendescribed. Most approaches are aimed at on-line monitor-ing, but considering the capillary storage off-line analysiscould also be attractive [62], because in that case, sloweranalytical procedures that maximize analytical sensitivityare easier.

Possible applicationsThe importance of continuouslymeasuring the actual levelsof a compound under non-steady state conditions has beenillustrated elegantly with a series of experiments usingMF.Here, MF probes were placed in the aorta and the jugularvein of an anaesthetized rat and glucose and lactate weremeasured simultaneously every minute in both blood lines,which resulted in more than 1000 data points per workingday [28]. This allowed for mathematical modelling of theresults [53]. These studies have shown that the transport oflactate is concentration driven, both into, as well as out ofthe brain. Presumably bi-directional lactate transportersappear to be not much different from those that have beenwell established for muscle [28,53]. A similar approach hasallowed monitoring of the release of cardiac lactate duringexperimental infarction in anaesthetized pigs [63]. Inanother study, a wearable device has been used to monitorsubcutaneous glucose in human volunteers [64,65] with theaim of monitoring glucose metabolism in freely activehumans, which might be useful, for example, in the controlof diabetes. The usefulness of the storage capillary has beendemonstrated in chicken and cows as discussed above[26,40].Here, venousMFprobeswere connected to a storagecapillary and a vacuum pump was attached under the wingof freely moving broiler chickens, to monitor blood levels oflactate and glucose. During a period of about 8 h, venousultrafiltrates were collected and analyzed later using aparallel set-up of micro-reactors for lactate and glucosedetermination. The level of both these analytes increasedconcomitantly with feeding [40]. MF also has been used toassess the individual stress sensitivity of cows using probesplaced in the udder vein, and pumps and storage capillaryattached in a holder placed near the udder. Some cows had a

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persistently low level of cortisol, whereas others, apparentlymore stressed, exhibited transiently increased hormonelevels [26].

Recently slow MD has been used in humans to inves-tigate the release of interleukins (ILs) during UV-inducedinflammation of the skin [66]. The results have indicatedthat local tissue injury induces the direct release of pre-formed IL-6 and IL-8 by keratinocytes and fibroblasts. Bycontrast, the cytokine response to UV irradiation is drivenmainly by T-helper cell TH1- and TH2-related cytokines,which is finally terminated by TH2 cytokines IL-4 and IL-10. The authors have concluded that the combination ofdermalMD and proteinmicroarray offers a powerful tool toanalyze in situ and over time the complex and rapidlychanging interstitial protein milieu during cutaneousinflammatory responses.

The versatility of ultraslow MF has been demonstratedin a number of studies that have aimed to characterizespecific proteins or protein profiles [27,46–48,67–69].Filtrates have been obtained in vivo from mouse or humansaliva and from various tissues, including tumours, skinwounds and subcutaneous tissue. MF devices also havebeen used successfully in vivo to determine the proteincomposition of blood plasma, tumour secretomes, salivaand wounded skin [27,46–48,67–69]. The subcutaneousset-up of Huang and co-workers [46–48,68,69] is shownin Figure 2. With this set-up, the extracellular release of b-thymosin during skin wound healing was monitored inanaesthetized mice [48]. The capillary MF probe wasimplanted by inserting a sterile 22-gauge needle into thewounded area of the skin. Approximately 1 ml of fluid washarvested from wounded and unwounded (normal) skin

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after a 3 h collection. The wound fluid was digested withtrypsin and the complex mixture of peptides was subjectedto matrix-assisted laser desorption ionization/time-of-flight mass spectrometry analysis. This study is believedto be the first to demonstrate the coexistence of thymosinsb-4 and b-10 in wound fluids.

Conclusions and future prospectsThanks to the recent developments of sensitive analyticaltechniques and methods to handle nanolitre samples,ultraslow MD and MF have now become realistic optionsfor in-line, on-line and off-line monitoring. The issuesconsidered here are subjected to a SWOT (strengths, weak-nesses, opportunities and threats) analysis, as summar-ized Table 2. Although in vitro applications currentlyremain sparse, the available probes could be used easilyin small reaction vessels with volumes as small as 25 ml.Ultraslow sampling in pharmacokinetics studies has justbegun to be exploited and on-site monitoring has beenfacilitated considerably by the introduction of MQMD,which allows convenient sample handling and couplingwith LC and MS analytical devices for on-line monitoring.Moreover, capillary storage of sub-microlitre samplesallows the analysis of time profiles not only in the labora-tory, but also in resource-poor settings.

In essence, many of the above-described MD and MFtechnologies are miniaturized versions of conventional MDand MF approaches. A major vision for the field is incontinuing the progress that has already been made incombining miniaturized and mass-produced microfluidicanalytical systems [55,59,70,71], with ultraslow MD andMF. This will eventually enable the realization of fullyautomatic devices that allow precise monitoring of ana-lytes that can be applied to on-site monitoring and atlocations without laboratory facilities.

Disclosure statementK.D. Huinink is currently employed by Brainlink BV, TheNetherlands, with commercial interest in the MQMD tech-nique. J. Korf and G.A. Posthuma-Trumpie declare noconflict of interest.

AcknowledgementsWork by the authors is supported by the Dutch Technology Foundation(Stichting Technische Wetenschappen, STW), Applied Science Division ofthe Netherlands Organization for Scientific Research (NWO) and theTechnology Program of the Ministry of Economic Affairs, grant numbersGPG 06038 and GGN 04680), and by the Commission of the EuropeanCommunities, Director General for DG XII Science, Research andDevelopment, Biomed 2 Program PL-972726.

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