1 23

Microchimica ActaAn International Journal onMicro and Trace Chemistry ISSN 0026-3672Volume 172Combined 1-2 Microchim Acta (2010)172:225-232DOI 10.1007/s00604-010-0459-1

Towards a monolithically integratedmicrosystem based on the greentape ceramics technology forspectrophotometric measurements.Determination of chromium (VI) in water

1 23

Your article is protected by copyright and

all rights are held exclusively by Springer-

Verlag. This e-offprint is for personal use only

and shall not be self-archived in electronic

repositories. If you wish to self-archive your

work, please use the accepted author’s

version for posting to your own website or

your institution’s repository. You may further

deposit the accepted author’s version on a

funder’s repository at a funder’s request,

provided it is not made publicly available until

12 months after publication.

ORIGINAL PAPER

Towards a monolithically integrated microsystem basedon the green tape ceramics technology for spectrophotometricmeasurements. Determination of chromium (VI) in water

Ricardo Alves-Segundo & Núria Ibañez-Garcia &

Mireia Baeza & Mar Puyol & Julián Alonso-Chamarro

Received: 20 May 2010 /Accepted: 5 September 2010 /Published online: 16 October 2010# Springer-Verlag 2010

Abstract A general procedure is presented for the fabricationof miniaturized continuous flow analytical microsystemsbased on photometric detection using the low temperatureco-fired ceramics technology. Optical elements such as lightemitting diodes and photodiodes (all in the size of a fewhundred micrometers) are integrated by means of an off-chipapproach. A simple procedure is demonstrated to integrate aglass window after the ceramic sintering in order to minimizethe decrease in sensitivity due to the reduction of the opticalpath length when scaling down. A flow cell with a shape ofa bubble has been used to increase the area of the lightbeam. The device is robust, affordable, and small-sized. Itenables absorbance measurements to be performed in-situor for continuous monitoring of environmental samples.Specifically, a microsystem is introduced for colorimetricdetermination of chromium (VI) ion in waters based on thediphenylcarbazide reagent as a model. Under the opti-mized conditions, a linear response is obtained for theconcentration range from 0.1 to 20 mg L−1, with adetection limit of 50 μg L−1.

Keywords Low-temperature co-fired ceramics .

Microanalyzer . Photometric detection . Chromium (VI) .

Diphenylcarbazide

Introduction

Since the beginning of the 90’s, when Manz et al. [1]proposed the exploitation of the concept miniaturization-integration to develop miniaturized analytical instrumenta-tion, capable of integrating the required steps to perform achemical analysis, an enormous effort has been made toachieve this goal [2–4]. The main materials employed forthe procurement of such structures are silicon, glass orpolymers with their associated techniques. However, silicon[5, 6] and glass are limited to obtain bidimensionalstructures and involve complex fabrication processes andexpensive clean room facilities, which obviously haverepercussions on the prototyping rate and the unitary costs.Moreover, it is not possible to monolithically integrateelectronic circuits and fluidics in the same device. Poly-meric materials have been gaining importance during thelast years as they allow obtaining disposable devices in arapid way [7]. In addition, it is possible to construct three-dimensional structures by means of a multilayered lamina-tion [8–10]. However, they also show some drawbacks as apoor chemical resistance, limited thermal stability and adifficult sealing of the microchannels [11].

The ideal fabrication technology for the development ofminiaturized analytical systems would be the one thatcombine the versatility of a multilayered fabrication withother features such as the rapid prototyping and thepossibility of mass production at low cost and all that,avoiding the use of clean room facilities. The lowtemperature co-fired ceramics technology (LTCC) joins all

Electronic supplementary material The online version of this article(doi:10.1007/s00604-010-0459-1) contains supplementary material,which is available to authorized users.

R. Alves-SegundoEscola Superior de Biotecnologia,Universidade Católica Portuguesa, Centro Regional do Porto,Campus da Asprela,4200-072 Porto, Portugal

N. Ibañez-Garcia :M. Baeza :M. Puyol :J. Alonso-Chamarro (*)Grup de Sensors i Biosensors, Departament de Química,Facultat de Ciències, Edifici C-Nord,Universitat Autónoma de Barcelona,08193 Bellaterra (Cerdanyola del Vallès), Spaine-mail: julian.alonso@uab.es

Microchim Acta (2011) 172:225–232DOI 10.1007/s00604-010-0459-1

Author's personal copy

these requirements [12]. Its main feature is that allows thefabrication of microfluidic platforms of complex structures,which show a hermetic sealing of the channels after asinterization step by means of an easy multilayeredmethodology. Moreover, it permits to integrate fluidic,mechanical and electronic components in the same ceramicsubstrate [13]. All these characteristics provide greatversatility and make available the integration of therequired different unitary operations of the analyticalprocedure, such as the sample pre-treatment or the analytedetection [14]. Other outstanding features are the rapidity,simplicity and low cost of the prototyping process, whichmake agile the optimization of the design of the structuresand its easy adjustment to each raised analytical applica-tion. Finally, the physico-chemical properties of the ceramicmaterial are especially adequate when robust autonomousmicroanalyzers are to be used in hard environmentalconditions [15].

The capability of the LTCC technology to the design ofanalytical microsystems, which monolithically integratedifferent types of electrochemical and photometric detec-tors, has been previously demonstrated [16–18]. Althoughabsorbance measurements are the most widely employedoptical detection methods in routine analytical chemistry,their application in ceramic microfluidic systems is scarce[19–21] due to the derived negative effect of scaling downon the sensitivity. However, a proper integration of opticalelements on the microfluidic platform can help to minimizethis problem. Recently, Nuriman et al. [22] classified theoptical detection approaches described for microfluidicsystems in two main groups: those where macroopticalcomponents were used in an “off-chip approach” and the socalled “on-chip approach”, where microoptical componentsare monolithically integrated into de microfluidic platform.Up to now, the second group is at the early developingstage due to the inherent difficulties of the monolithicintegration of waveguides, detectors and emitters on thesame substrate [23, 24] than the microfluidic platform.

On the other hand, microfluidic systems applied tobiological or environmental fields tend to exploit other keyfeatures of miniaturization like automation, robustness orportability [25–27]. By an off-chip approach, low-cost andsmall optical elements, such as light-emitting diodes(LEDs) and photodiodes [7, 20, 21], can be used if thedimensions of the microfluidic channels are in the range ofhundred instead of tens of microns. In this applicationniche, the LTCC technology provides almost all therequired features.

On the contrary, green ceramic tapes lack of materialtransparency. Golonka et al. [28] developed a LTCCmicrosystem incorporating a Z-shape flow cell, which isirradiated with optical fibers to avoid this problem, butunsatisfactory results in terms of level of noise, signal

repeatability and drift are observed. Even though thisapproach could be feasible, the structural weakness of theoptical fibers reduces the robustness of the whole device.Other possibility is the integration of optical windows in themicrofluidic platforms. Although transparent materials likesapphire, with a melting point higher than the ceramicmaterial, can be monolithically integrated, a simpler andless expensive procedure to integrate a glass window afterthe sinterization process is employed in this work. Tominimize sensitivity and limit of detection (LOD) problemsrelated to the reduction of the optical path length in themicrosystems, a bubble-configuration flow cell is used [29,30]. It allows increasing the sampled light beam area in orderto compensate the path length reduction. Additionally,exploiting the multilayer approach, further improvements insensitivity can be achieved when needed, by simply addingmore ceramic layers to increase the microchannel depth.

In this work, the versatility of the approach is demon-strated by developing a robust continuous flow analyticalLTCC microsystem for on-line monitoring based onabsorbance measurements. The level of miniaturization issettled in order to achieve some characteristics as easyhandling and operation, simplified connection between themicrosystem and the real world and, robustness in front ofcomplex sample matrices and severe environmental con-ditions. The developed device can be applied to a widevariety of colorimetric determinations of analytes withoptical properties or other products obtained on chemicalreactions. To illustrate its operational features, a continuousflow analytical microsystem has been evaluated as a modelreaction for the photometric determination of chromium(VI) in water. Cr (VI) is generally produced by industrialprocesses and its known potential risks as carcinogen hashamper its determination in industrial effluents and surfacewaters. Although the European Union has set the maximumlevel of chromium in drinking water to 50 μg L–1

(according to the European Community Directive 80/778/EEC, L229/20, D48) [31], concentrations of chromium (VI)compounds exceeding this value are still often found ingroundwater near industrial sites because they werecomponents of dyes and paints and were widely used forleather tanning [32].

Continuous on-line measurements might help to rapidlyact in front of a sudden contamination episode, especially inriver courses near water treatment plants for humanconsumption. Autonomous miniaturized analytical systemscan be used to obtain real time information, whenintegrated in automatic alert stations, although this can beless precise. In this sense, different compact and portable Cr(VI) analyzers, which are still in a developing phase, can befound in the literature [33–37]. In the present work, the useof the reference method based on 1,5-Diphenylcarbazide(DPC) colorimetric reagent for Cr (VI) has been selected

226 R. Alves-Segundo et al.

Author's personal copy

[38] for an on-line application. The intense pink/violet colorof the formed product as a result of the reaction is measuredat 543 nm.

Experimental

Materials and equipment

Dupont 951 green ceramic tapes are used as substrate forthe fabrication of the microanalyzer. A laser (Protolaser,LPKF, Germany, www.lpkf.com) and a CNC (ComputerNumerically Controlled) machine (ProtoMat, LPKF,Germany, www.lpkfusa.com) are used for green tapesmachining. A thermo-compression press (Francisco Camps,Granollers, Spain) is used for ceramic sheet alignment andlamination. Finally, the devices are sintered in a program-mable box furnace (Carbolite, Afora, Spain, www.afora.es).To perform optical measurements a mechanized cavitydefining a flow cell with a bubble configuration is coveredwith two transparent glass windows, which are glued to theceramic surface using polydimethylsiloxane (PDMS) assealant, purchased from Dow Corning (Midland, Michigan,US, www.dowcorning.com).

A hybrid system using meso-scale actuators for fluidhandling is designed. The continuous flow system setupconsists of a peristaltic pump (Minipuls 3, Gilson,Wisconsin, US, www.gilson.com) equipped with 1.14 mminternal diameter silicon tubing (Ismatec, Zürich, Switzerland,www.ismatec.com) and a six-port injection valve (HamiltonMVP, Reno, US, www.hamiltoncompany.com). 0.8 mminternal diameter Teflon tubing (Scharlab, S.L., Cambridge,England, www.scharlab.com) are used to connect theexternal elements with the microsystem. A poly-methylmethacrylate (PMMA) T-shaped external structure is usedfor the optimization of the composition of the reagentsolution. However, miniaturized alternative actuators arebeing studied, in order to improve portability.

The detection system consists in a 565 nm emitting greenLED (L-483 GDT, KingBright, Taiwan, www.farnell.com/es)and a Hamamatsu photodetector (model S1337-66BQ)(Hamamatsu Corporation, Parque Tecnológico del Vallés,Cerdanyola, Spain, sales.hamamatsu.com), connected to aLabview data acquisition card through two exit lines(potential and reference). The signal acquisition software iscustom made to this detection system.

Fabrication of the microanalyzer

The general fabrication process [14] is based on amultilayer approach, using layers of the 951AX green tapeceramics (thickness of 245 μm) as a basic building block.The design of the microfluidic platform, including the

cavities for the optical flow-cell, is developed with CADsoftware. Figure 1 (a–e) shows the required layers toconfigure the final device (F). A total amount of eightlayers (4×A, 1×B, 1×C, 1×D and 1×E) is used. A is theupper layer for external flow connections and the sealedglass window for optical measurements. Four layers arerequired (4×A) for the insertion and sealing of flowconnectors (Fig. 1g). B is the following layer with themechanization of the internal connections to flow channelsand the optical flow cell of a bubble shape. C is the thirdlayer with the mechanized flow channels and optical flowcell. In D layer the optical flow cell is again mechanized toassure enough optical path and E is the lower layer to attachanother glass window (Fig. 1h). In order to compensatethe shrinkage caused by the sintering process (12.3% inthe x-y axis and 15% in the z axis), the cavities designedto embed the flow connectors and optical flow cell areslightly bigger. Taking into account the shrinkagesuffered by the ceramics the dimensions of the channelsare 0.85 mm wide and 0.2 mm height after firing. Thecavity designed to integrate the optical windows have thefollowing dimensions: maximum wide 4.3 mm × maxi-mum long 6.8 mm × height 0.6 mm. After the firingprocess, final effective reactor and optical flow cellvolumes are 55.12 μL and 26.68 μL, respectively. Thusthe total internal volume of the microsystem is 81.80 μLand the optical path length is 0.6 mm.

All the layers are individually mechanized with a lasermachine. After that, they are aligned in the following order(A–E) by means of four fiducials determined during themachining process, and laminated with a home-madethermo-compression press. The laminated block is cut witha CNC machine in order to separate the different micro-devices and then, they are sintered in a programmable oven.The external dimensions of each device are 2.7×2.7×0.15 cm after firing. Finally, the fluidic connectors areglued onto the LTCC inlet/outlet ports with standard epoxyglue (Fig. 1g). To obtain a transparent radiation areathrough the ceramic platform to perform optical measure-ments, the previous mechanized cavity is covered with twotransparent glass windows. They are glued to the ceramicsurface using PDMS as a sealant (see Fig. 1h). A 5 mmdiameter LED allows irradiating all the transparent areadefined by the glass windows by the upper side. Thephotodiode is located at the other side by covering approx-imately a 66% effective area of the transparent window. Thisconfiguration allows a sensitivity improvement.

Reagents, standards and samples

All the reagents used are of analytical grade and allsolutions are prepared using deionized water from a Milli-Q system (Millipore, Billerica, MA, USA). Chromium

Towards a monolithically integrated microsystem 227

Author's personal copy

stock solution (100 mg L−1) is prepared by weighing outand dissolution of K2Cr2O7 (99%, Sigma-Aldrich, St.Louis, USA) in deionized water and stored in an opaquebottle. Cr (VI) (ranging from 0.05 to 50 mg L−1) standardsolutions are prepared daily by appropriate dilution.

The solution of the complexing reagent is prepared bydirect weighing out 1,5-diphenylcarbazide (DPC) (Doesder,Barcelona). The composition of the final acidified (0.1 MH2SO4) colorimetric reagent solution is 1.25·10−3 M DPCwith a 10% of ethanol purchased from Panreac (Barcelona,Spain). The reagent is stable for one month when stored ina dark bottle at 4°C. Sulphuric acid solutions are preparedfrom concentrated sulphuric acid (Sigma-Aldrich, St. Louis,USA).

Superficial waters from Besos river (Barcelona, Spain) aredoped with increasing quantities of Cr (VI) by appropriatedilution of a 100 mg L−1 chromium stock solution.

Experimental procedure

A schematic representation of the continuous flow systemset-up is show in Fig. 2a. During the optimization process,H2SO4 and DPC solutions have been mixed using a T-shaped PMMA structure out of the microsystem and the

resulting solution has been carried to the LTCC device,where it mixes with the standards that are inserted in acarrier composed by deionized water using a six-portrotatory injection valve. An intensive pink/violet complex(Cr (VI) -DPC) is formed and detected.

A black PMMA (Fig. 2b) support is employed to assemblethe light emitting diode, the photodiode (Fig. 2c) and theassociated electronics for the off-chip integration, whichpermits an in-line optical measurement.

Results and discussion

The main goal of the present work is to demonstrate thepossibility to develop simple photometric microanalyzersbased on absorbance measurements, which monolithicallyintegrate a microfluidic platform and a detection flow cellusing the LTCC technology without a sensitivity loss. Asmentioned above, the major concern about the applicationof optical detection in microfluidics is related to the effectof scaling down on sensitivity. The reduction of the opticalpath length and the number of analyte molecules in thesample volume worsen the detection limit, especially in theabsorbance mode.

Fig. 1 LTCC miniaturizedphotometric analyzer. Innermicrosystem architecture (CADdesign); a-e: layout of thedifferent layers and F: finaldevice with all the overlappedlayers. G: external fluidicconnectors, a) teflon tube; b)flexible silicon tube; c) metallicconnector; d) epoxy adhesivesealant; e) ceramic microsystem.H: optical flow cell; f) lightbeam; g) glass windows; h) lightpath length. I: photograph of thewhole microsystem

228 R. Alves-Segundo et al.

Author's personal copy

Microsystem optimization

Initially, the optimization of the chemical and the hydrody-namic parameters of the microanalyzer have been per-formed in order to establish the better conditions to obtainthe best analytical response: higher sensitivity and lowerdetection limit for the analyte detection. These experimentalvariables are the sample injection volume, the flow rate, thesulphuric acid concentration and the DPC concentration.For all experiments, 0.1 and 2.0 mg L−1 Cr (VI) standardshave been used in order to evaluate the system response athigh and low concentration levels. The injection volumehas been varied from 50 to 1500 μL.

According to the obtained results (shown as supplemen-tary data), an injection volume of 500 μL is chosen as theoptimum. Higher sample volumes (V>500 μL) do notsignificantly increase the obtained signal for 0.5 and2.0 mg L−1 and the sample throughput is drasticallyreduced. For lower chromium concentrations the signaldoes not increase for volumes higher than 180 μL.However, at high concentrations of sample, a rapid loss ofsensitivity is observed at low injection volumes, thusworsening the detection limit.

The effect of the total flow rate has been evaluated in therange between 0.39 and 1.30 mL min−1. A constant 1:1relation between the flow rate of the carrier and the reagentsolutions has been maintained all through the study. Majordifferences are observed with the flow rate increase forconcentrated standards. The reaction time is not enoughlong using the higher flow rate (1.3 mL min−1) and a

reduction of a 25% of the analytical signal is noticeable inrelation with the lower flow rate (0.39 mL min−1). Thereaction time is constant at all flow rates but at the highestflow rate the reaction time is too short to assure an optimalanalytical signal and consequently the peak height is lower.An optimum value of 0.66 mL min−1 has been chosen as acompromise in order to maintain the sampling rate, assurethe necessary reaction time and allow the detection of lowCr (VI) concentrations. At this flow rate the reduction ofthe analytical signal, in relation with the lower flow ratestudied (0.39 mL min−1), is of 3 and 8% for 0.1 and2 mg L−1, respectively.

To enhance the obtained sensitivity with the micro-system, the composition of the colorimetric reagent solutionhas been also optimized. As the reaction between Cr (VI)and DPC occurs in a sulphuric acid medium, the influenceof the concentration of both reagents has been evaluated.During this study and in order to simplify the experimentalset-up, the channel corresponding to the reagent solutionhas been split in two (see Fig. 2a). In this way, anindividualized optimization can be performed. The concen-tration of the sulphuric acid solution (A1 in Fig. 2a) isvaried from 5 10−3 to 4 10−1 M. Results show that lowerconcentrations of sulphuric acid do not to provide ameasurable signal for the lowest Cr (VI) concentration(0.1 mg L−1). A concentration of 2 10−1 M sulphuric acid ischosen as the optimum because higher values do notsignificantly improve the obtained response and as adrawback, this can bring in additional problems relatedwith a higher back pressure due to the reagent viscosity and

c 1

2

3

4

b

aFig. 2 Continuous flow micro-system set-up. a) Experimentalmanifold: A, acidified DPCreagent solution; A1 H2SO4 so-lution; A2, DPC reagent solu-tion; B, deionized water; S,sample; P, peristaltic pump; V,six-port injection valve. b) Pro-tective PMMA black support forthe external optical components.c) Optical detection set-up: (1)LED; (2) PMMA support; (3)Photodetector and associatedelectronics; (4) DB9 (RS232)computer connector

Towards a monolithically integrated microsystem 229

Author's personal copy

alterations in the detector response due to refractive indexvariations [39]. With these experimental conditions theefficiency of the passive mixer was optimal and a completemixing in the microsystem is achieved.

For the optimization of the DPC reagent solution (A2 inFig. 2a), concentrations ranging from 1 10−3 to 5 10−3 Mhave been evaluated. For the tested levels of Cr (VI)concentration, the higher the concentration of DPC is, thebetter sensitivity is obtained. Since DPC concentrationshigher than 5 10−3 M give solubility problems, thisconcentration is chosen as the optimum.

Microsystem characterization

After the optimization of the system and using the selectedexperimental conditions, the analytical features of thedeveloped microsystem have been determined from succes-sive calibrations performed in the course of six non-consecutive days. The analytical frequency was 20 sampleshour−1 and the reagent consumption is of 1.98 mL sample−1.

The detection limit (LOD) has been calculated accordingto IUPAC [40]. The minimum detectable signal has beenestimated from a 10 blank series and its standard deviation,and it has been calculated as SignalLOD=Sb + k·sb (whereSb is the average of the blank signals, sb is the standarddeviation of the blank signals and k=3) by interpolatingthis value in the calibration curve. The minimum concen-tration detectable was 0.05 mg L−1.

The repeatability of the system has been estimated bymeans of the relative standard deviation obtained from14 replicates of a 2 mg L−1 Cr (VI) standard. The averagepeak height and RSD are 0.727±0.008 mV and 2.06%,respectively.

Reproducibility has been determined by comparison ofthe slopes of calibration curves performed on different days(n=6). The ANOVA performed show that there is nosignificant differences between calibration curves with a95% confidence since Fcal=3.7<Ftab=9.6.

An example of the obtained analytical signal can be seenin Fig. 3. For n=5 and at 95% significance, the linear

Time (s)

0 1000 2000 3000 4000 5000 6000

Pot

entia

l (m

V)

-10

-8

-6

-4

-2

0

Time (s)0 200 400 600 800 1000

Pot

entia

l (m

V)

-0.08

-0.06

-0.04

-0.02

0.00

0.1 mg L-1

1mg L-1

2 mg L-1

5 mg L-1

20 mg L-1

30 mg L-1

50 mg L-1

Fig. 3 Response signalobtained, by injecting Cr (VI)from 0.1 to 50 mg L–1, using theoptimal experimental conditions

Water sample turbidity (ntu*) Theoretical (mg L−1) Found (mg L−1) Difference Recovery (%)

10 0.99 0.93±0.03 −0.06 93.9

1.96 2.06±0.03 0.10 105.1

4.75 4.82±0.09 0.07 101.5

20 0.99 0.96±0.05 −0.03 97.0

1.96 2.09±0.02 0.13 106.6

4.75 4.72±0.08 −0.03 99.4

30 0.99 1.02±0.05 0.03 103.0

1.96 2.073±0.006 0.11 105.8

4.75 4.74±0.10 −0.11 99.8

Table 1 Cr (VI) recovery datain real samples spiked with in-creasing quantities of chromium.Error was estimated as CI95% forn=3 determinations, CI95% ¼Sn�1 � tn�1 n

12

.

* Turbidity units

230 R. Alves-Segundo et al.

Author's personal copy

response is Abs=0.2 (±0.3)+0.27 (±0.04) [Cr (VI)] (r2=0.995, 95% confidence) from 0.1 to 20 mg L−1. The linearresponse range is one concentration decade higher thanother previous published works (0.3–1.5 mg L−1) [37].

The system has been tested with spiked real samplescoming from Besos River in Catalonia, Spain. Threesamples with different turbidity have been analyzed intriplicate after the addition of different amounts of a stocksolution to obtain the final concentrations of 0.99, 1.96and 4.75 mg L−1 of Cr (VI). The efficiency of the methodhas been determined by the difference between thetheoretical value and the obtained one and, the % ofrecovery of the added Cr (VI). As can be seen on the resultssummarized in Table 1, a high recovery was obtained for allsamples.

Conclusions

The feasibility of using the LTCC technology for the designof analytical microsystems based on absorbance measure-ments has been demonstrated. The problems related withthe lack of transparency of the ceramic material have beenavoided by the integration of an optical flow cell with twoglued glass windows. Moreover, low-cost and small opticalelements, such as LEDs and photodiodes have been easilyintegrated by means of an off-chip approach. On the otherhand, sensitivity problems are minimized by the use of abubble-configuration flow cell and its coupling to the opticalsystem (led/photodiode). The bubble shape-configurationflow cell minimizes the possible dead volumes in front of theoptical system and shows a considerably higher radiationinteraction area than a spherical or a square configuration.Relative orientations of the led/photodiode improve collec-tion of light. The obtained LOD and sensitivity areequivalent to previous works, while the linear range ishigher in one concentration decade.

The LTCC multilayer approach also permits to obtainfurther sensitivity improvements, with the design of otherflow cell configurations without the alteration the micro-channel geometry at the detection cell, which are understudy. The present device is a hybrid flow system, usingminiaturized flow system platforms and discrete actuators.Further developments are focused to a more fully integratedmicrosystem to allow exploiting some key features ofminiaturization such as automation, robustness or portabil-ity to field applications. Alternative miniaturized actuatorsare being employed, in order to improve portability.

Acknowledgements This work was supported by the Spanish MECthrough the CICYT projects TEC2006-13907-C04-04/MIC and CIT-310200-2007-29. The authors are thankful to Oriol Ymbern for thefabrication the PMMA support, to Cynthia S. Martínez-Cisneros forthe development of the data acquisition software and to the Grupo de

Tecnologías Fotónicas from the University of Zaragoza for thefabrication of the optical detection system.

References

1. Manz A, Graber N, Widmer HM (1990) Miniaturized totalchemical analysis systems: a novel concept for chemical sensing.Sens Actuators B: Chemical 1:244–248

2. Reyes DR, Iossifidis D, Auroux PA, Manz A (2002) Micro totalanalysis systems. 1. Introduction, theory, and technology. AnalChem 74:2623–2636

3. Auroux PA, Iossifidis D, Reyes DR, Manz A (2002) Micro totalanalysis systems. 2. Analytical standard operations and applications.Anal Chem 74:2637–2652

4. West J, Becker M, Tombrink S, Manz A (2008) Micro totalanalysis systems: latest achievements. Anal Chem 80:4403–4419

5. Baeza MM, Ibanez-Garcia N, Baucells J, Bartrolí J, Alonso J(2006) Microflow injection system based on multicommutationtechnique for nitrite determination in wastewaters. Analyst131:1119–1115

6. Pumera M, Merkoçi A, Alegret S (2006) New materials forelectrochemical sensing VII. Microfluidic chip platforms. TrendsAnal Chem 25:219–235

7. Ramírez-García S, Baeza M, O’Toole M, Wu Y, Lalor J, WallaceGG, Diamond D (2008) Towards the development of a fullyintegrated polymeric microfluidic platform for environmentalanalysis. Talanta 77:463–467

8. Liu Y, Lu H, Zhong W, Song P, Kong J, Yang P, Girault HH, Liu B(2006) Multilayer-assembled microchip for enzyme immobilizationas reactor toward low-level protein identification. Anal Chem78:801–809

9. Moss ED, Han A, Frazier AB (2007) A fabrication technology formulti-layer polymer-based microsytems with integrated fluidicand electrical functionality. Sens Actuators B: Chemical 121:689–697

10. Fuentes HV, Woolley AT (2008) Phase-changing sacrificial layerfabrication of multilayer polymer microfluidic devices. AnalChem 80:333–339

11. Muck A, Svatos A (2007) Chemical modification of polymermicrochip devices. Talanta 74:333–341

12. Gongora-Rubio MR, Espinoza-Vallejos P, Sola-Laguna L,Santiago-Avilés JJ (2001) Overview of low temperature co-firedceramics tape technology for meso-system technology (MsST).Sens Actuators A: Physical 89:222–241

13. Martínez-Cisneros CS, Ibañez-García N, Valdés F, Alonso J (2007)Miniaturized total analysis sytems: Integration of electronics andfluidics using low-temperature co-fired ceramics. Anal Chem79:8376–8380

14. Ibáñez-García N, Alonso J, Martínez-Cisneros CS, Valdés F(2008) Green-tape ceramics. New technological approach forintegrating electronics and fluidics in microsystems. Trends AnalChem 27:24–33

15. Diamond D, Sequeira M, Bowden M, Minogue E (2002) Towardsautonomous environmental monitoring systems. Talanta 56:353–363

16. Ibañez-García N, Machado Gonalves RD, Mendes da Rocha Z,Góngora-Rubio MR, Seabra AC, Alonso-Chamarro J (2006)LTCC meso-analytical system for chloride ion determination indrinking waters. Sens Actuators B: Chemical 181:67–72

17. Ibanez-Garcia N, Bautista-Mercader M, Mendes da Rocha Z, SeabraCA, Gongora-Rubio MR, Alonso-Chamarro J (2006) Continuousflow analytical microsystems based on low-temperature co-firedceramic technology. Integrated potentiometric detection based onsolvent polymeric ion-selective electrodes. Anal Chem 78:2985–2992

Towards a monolithically integrated microsystem 231

Author's personal copy

18. Llopis X, Ibáñez-García N, Alegret S, Alonso J (2007) Pesticidedetermination by enzymatic inhibition and amperometric detectionin a LTCC microsystem. Anal Chem 79:3662–3666

19. Golonka LJ, Zawada T, Radojewski RH, Stefanow M (2006)LTCC microfluidic system. Int J Appl Ceram Techno 3(2):150–156

20. Ibáñez-García N, Puyol M, Azevedo CM, Martínez-Cisneros CS,Villuendas F, Gongora-Rubio MR, Seabra AC, Alonso J (2008)Vortex configuration flow cell based on low-temperature cofiredceramics as a compact chemiluminescence microsystem. AnalChem 80:5320–5324

21. Baeza M, López C, Alonso J, López-Santín J, Alvaro G (2010)Ceramic microsystem incorporating a microreactor with immobi-lized biocatalyst for enzymatic spectrophotometric assays. AnalChem 82:1006–1011

22. Nuriman BK, Huskens J, Verboom W (2007) Optical sensingsystems for microfluidic devices: a review. Anal Chim Acta601:141–155

23. Van Overmeire S, Ottevaere H, Desmet G, Thienpont H (2008)Miniaturized detection system for fluorescence and absorbancemeasurements in chromatographic applications. IEEE J QuantumElectron 4:140–150

24. Mogensen KB, Kutter JP (2009) Optical detection in microfluidicsystems. Electrophoresis 30:S92–S100

25. Rodrigues ERGO, Lapa RAS (2009) Development of micro-flowdevice by direct-milling on poly(methyl methacrylate) substrateswith integrated optical detection. Microchim Acta 166:189–195

26. Xu Z-R, Wang X, Fan X-F (2010) An extrusion fluidic drivingmethod for continuos-flow polymerase chain reaction on amicrofluidic chip. Microchim Acta 168:71–78

27. Wang Y, Chen Z-Z, Li Q-L (2010) Microfluidic techniques fordynamic single-cell analysis. Microchim Acta 168:177–195

28. Golonka LJ, Roguszczak H, Zawada T, Radojewski J, GrabowskI, Chudy M, Dybko A, Brzozka Z, Stadnik D (2005) LTCC basedmicrofluidic system with optical detection. Sens Actuators B:Chemical 111–112:396–402

29. Liu S, Dasgupta PK (1993) A simple means to increaseabsorbance detection sensitivity in capillary zone electrophoresis.Anal Chim Acta 283:747–753

30. Xue Y, Yeung ES (1994) Characterization of band broadening incapillary electrophoresis due to nonuniform capillary geometries.Anal Chem 66:3575–3580

31. Vercoutera K, Cornelis R, Mees L, Quevauviller PH (1998)Certification of the contents of the chromium (III) and chromium(VI) species and total chromium in a lyophilised solution (CRM544). Analyst 123:965–969

32. Fantoni D, Brozzo G, Canepa M, Cipolli F, Marini L, Ottonello G,Zuccolini MV (2002) Natural hexavalent chromium in groundwatersinteracting with ophiolitic rocks. Environ Geol 42:871–882

33. Pires CK, Reis BF, Morales-Rubio A, de la Guardia M (2007)Speciation of chromium in natural waters by micropumpingmulticommutated light emitting diode photometer. Talanta72:1370–1377

34. Pressman MAS, Aldstadt JH (2005) A remote in situ monitorbased on continuous flow analysis for the quantitation of sub-micromolar levels of hexavalent chromium in natural waters. JEnviron Monit 7:809–813

35. Long XB, Miro M, Hansen EH (2005) An automatic micro-sequential injection bead injection Lab-on-Valve (mu SI-BI-LOV)assembly for speciation analysis of ultra trace levels of Cr(III) andCr(VI) incorporating on-line chemical reduction and employingdetection by electrothermal atomic absorption spectrometry(ETAAS). J Anal At Spectrom 20:1203–1211

36. Wang J, Wang JY, Lu JM, Tian B, MacDonald D, Olsen K (1999)Flow probe for in situ electrochemical monitoring of tracechromium. Analyst 124:349–352

37. Fonseca A, Raimundo IM Jr, Rohwedder JJR, Ferreira LOS (2007)Construction and evaluation of a flow injection micro-analyser basedon urethane-acrylate resin. Anal Chim Acta 603:159–166

38. Clesceri LS, Greenberg AE, Eaton AD (1995) Standard methodsfor the examination of water and wastewater, 20th edn. UnitedBook Press, Baltimore

39. Alonso-Chamarro J, Bartroli J, Barber R (1992) Sandwichtechniques in flow-injection analysis. Anal Chim Acta 261:219–223

40. Inczedy J, Lengyel T, Ure AM (1988) Compendium of analyticalnomenclature. Definitive rules 1997, 3rd edn. Blackwell Science,Oxford

232 R. Alves-Segundo et al.

Author's personal copy