IEEE SENSORS JOURNAL, VOL. 8, NO. 7, JULY 2008 1305

Optical PMMA Chip Suitable forMultianalyte Detection

Francesco Baldini, Adolfo Carloni, Ambra Giannetti, Andrea Mencaglia, Giampiero Porro, Lorena Tedeschi, andCosimo Trono

Abstract—A new optical platform for the interrogation of apolymethylmethacrylate (PMMA) multichannel array for chem-ical and biochemical parameters is described. It consists of aplastic chip formed by two pieces of PMMA properly shaped inorder to obtain different microchannels, 500 m in width and400 m in height. A fluorescent sensing layer is immobilized on theinternal wall of the microchannel. The emitted light travels alongthe thickness of the upper PMMA piece and it is detected with anoptical fiber connected with a spectrum analyzer. The anisotropyof the fluorescence emitted by the fluorophore immobilized at theliquid/plastic interface gives rise to preferential directions of theemitted fluorescence. The collection of the fluorescence at an angleof 50 implies an amplification of roughly one order of magnitudein the ratio between the fluorescence signal and the scattered light.

The optical platform was further characterized as a pH sensorand as potential chip for immunoassay. In the first case, thefluorescein dye is directly immobilized onto the internal wallof the channel and the fluorescence changes are measured as afunction of the pH of the flowing sample. In the second case, adirect antigen–antibody interaction is carried out. The mouse-IgGis covalently immobilized onto the internal wall of the channel andthe Cy5-labeled anti-mouse IgG is used for the specific interaction.

Index Terms—Fluorescence anisotropy, IgG, multichannelarray, optical sensor, pH, polymethylmethacrylate (PMMA) chip.

I. INTRODUCTION

A N OPTICAL biochip can be considered an array of biosen-sors that are individually monitored and are generally used

for the determination of multiple analytes [1] both in biomedicaland environmental field. Because of its miniaturization, low cost,and potential for large-scale automation, it can perform analysismore efficiently than currently available laboratory equipment.A distinction can be made between label-free systems [2]–[4], inwhich the interaction analyte/sensitive layer gives rise to a mod-ification of the optical signal due to the change of the refractiveindex of the layer deposited on the substrate, and label-basedsystems [5]–[8], in which fluorescent labeling is used to get an

Manuscript received August 1, 2007; revised March 6, 2008; accepted April19, 2008. Published July 16, 2008 (projected). This work was supported by theEuropean Community within the framework of the four-year EU funded projectunder IST priority, CAREMAN (HealthCARE by Biosensor Measurements andNetworking). The associate editor coordinating the review of this paper and ap-proving it for publication was Prof. Brian Culshaw.

F. Baldini, A. Carloni, A. Giannetti, A. Mencaglia, and C. Trono are with theInstitute of Applied Physics “Nello Carrara”, CNR, Via Madonna del Piano 10,50019 Sesto Fiorentino (FI) Italy (e-mail: f.baldini@ifac.cnr.it; a.carloni@ifac.cnr.it; a.giannetti@ifac.cnr.it; a.mencaglia@ifac.cnr.it; c.trono@ifac.cnr.it).

G. Porro is with Datamed S.r.L., Via Papa Giovanni XXIII 45, 20090 Rodano(MI), Italy (e-mail: giampiero.porro@datamedsrl.com)

L. Tedeschi is with the Institute of Clinical Physiology, CNR, Via Moruzzi 1,56 124, Pisa, Italy (e-mail: tedeschi@ifc.cnr.it)

Digital Object Identifier 10.1109/JSEN.2008.926964

optical signal depending on the investigated analyte. Both thesemethods present advantages and disadvantages. Direct opticaldetection can boast the capability of analysis without the labelsapplication; in fact, fluorescence-based biosensors require eithermultistep detection protocols or delicately balanced affinitiesof interacting biomolecules for displacement assays, causingsensor cross-sensitivity to nontarget analytes. However, formonitoring complex samples, the label-free methods continueto be susceptible to problems such as low sensitivity andincreased backgrounds due to nonspecific binding.

The five-year integrated project “health CARE by biosensorMeasurements and Networking” (CARE-MAN) has the aim todevelop an intelligent and fully automated optical diagnosticdevice on a modular technological system. It will combinesuccessful transduction principles, biochemical recognitionmethods and communication capabilities to allow a multiparameter measurement characterizing diseases defined bydoctors and needs in hospitals. Within this project, a multi-channel array for chemical and biochemical parameters, whichhas to be part of the modular diagnostic device, is under devel-opment and is here described.

The present paper is concerned with the characterization of asingle channel, using a single source and a single detector, withthe purpose of demonstrating the efficiency of the system.

It consists of a plastic chip formed by two pieces of poly-methylmethacrylate (PMMA) opportunely shaped in order toobtain several flow channels. Light from a laser or a LED isused to excite the fluorescent sensing layer immobilized on theinternal wall of the channel. The emitted light travels along thethickness of the channel and is detected with an optical fiberconnected with a photodetector.

It is well-known that the emission of electric dipoles, such asthe excited fluorophores, is anisotropic when the distance froma dielectric interface is small or comparable with the emittedwavelength [9], [10]. Therefore, the optical chip was charac-terized in order to optimize the fluorescence collection, consid-ering the anisotropy of the fluorescence emission of the fluo-rophore at the plastic/liquid interface.

The preferential direction of emission was determined exper-imentally and the potentiality of the optical chip as chemicalsensor was investigated by:

1) immobilizing a pH indicator, fluorescein, in the PMMAchannel and evaluating the feasibility of a pH sensor withthis system;

2) immobilizing mouse-IgG that directly interact with theCy5-labeled anti-mouse-IgG and evaluating the capabilityof this system as a biochip.

1530-437X/$25.00 © 2008 IEEE

1306 IEEE SENSORS JOURNAL, VOL. 8, NO. 7, JULY 2008

Fig. 1. (a) Optical chip: longitudinal cross section. (b) Optical chip: transversal cross section.

II. EXPERIMENTAL

A. Reagents

All the following chemicals, of analytical reagent grade,were purchased at Sigma: fluorescein isothiocyanate (FITC),ethanol, sodium chloride (NaCl), disodium hydrogen phosphate(Na HPO ), potassium chloride (KCl), morpholinesulfonicacid monoidrato (MES), citric acid, disodium hydrogen phos-phate dodecahydrate (Na HPO 12 H O).

Eudragit RL 100 and Eudragit L 100 were purchased at De-gussa, Röhm Pharma Polymers.

Mouse-IgG and Cy5-labeled anti-mouse-IgG were purchasedat Zymed Laboratories, Invitrogen Immunodetection.

1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydro-chloride (EDC) and N-hydroxysuccinimide (NHS) were pur-chased at Pierce.

B. The Optical Chip

The optical chip consists of four channels obtained by com-bining two self-made PMMA pieces: the lower part which in-cludes the four microchannels and the inlet and outlet for thefluidic, and the upper part, where the sensing layer is immobi-lized. The dimensions of a single flow-channel are: 0.5 mm inwidth, 0.4 mm in height and 18 mm in length.

The longitudinal and transversal cross-sections of the flowcell are shown in Fig. 1(a) and (b), respectively. A photo ofthe realized prototype is shown in Fig. 2. The fluorophore im-mobilized on the lower surface of the cover is in contact withthe fluid under investigation. The excitation radiation is perpen-dicular with respect to the flow direction, and a fraction of theemitted fluorescence travels along the thickness of the PMMAupper piece up to its end-face, where it is collected by means ofa multimode optical fiber (200 m core diameter) coupled witha GRIN lens (SELFOC Microlens, 1/4 pitch, 1.8 mm diameter).The particular profile of the cover with the presence of air gaps,as shown in Fig. 1(b), allows a physical separation of the fluo-rescent signal coming from the different channels, so that eachchannel is optically separated from the others. The presence ofthe inlet and outlet for the fluidics on the bottom of the struc-ture facilitates the monitoring of the fluorescence which travelsinside the plastic cap along the same direction of the flow.

Fig. 2. Photo of the prototype of the PMMA flow cell.

As the fluorescein has an absorption peak near 490 nm, theexcitation source was a LED with emission peak at 488 nmfiltered by means of a bandpass filter at 488 nm (Andover488FS10-12.5, 10 nm FWHM).

The Cy5-labeled anti-mouse-IgG is characterized by anabsorption and emission peaks centered at 650 and 670 nm,respectively; therefore, a laser diode (Hitachi HL6314MG)with emission peak at 635 nm and 3 mW optical power is usedas excitation source and the collected fluorescence is filteredwith an high-pass filter (Thorlabs FEL0650, cut-on wave-length: 650 nm). The choice of the source at 635 nm, wherethe excitation efficiency is roughly 65%, follows the criterionof achieving a good separation between the used excitation andemission wavelengths.

In both cases, the signal collected by the fiber through theGRIN lens was detected and analyzed by means of an OceanOptics S2000 optical spectrum analyzer.

The optical chip is connected to a peristaltic pump(Minipuls3-Gilson) by means of PVC tubing (Gilson, i.d.0.76 mm). The flow rate used in all the experiments is30 l/min.

BALDINI et al.: OPTICAL PMMA CHIP SUITABLE FOR MULTIANALYTE DETECTION 1307

Fig. 3. (a) Fluorescence emitted at 539 nm as a function of the angle between the optical fiber and the flow directions. (b) Ratio of the fluorescence and theexcitation as a function of the angle between the optical fiber and the flow directions.

C. Preparation of the Sensing Layers and of the SampleSolution

For the realization of the pH sensor, FITC was immobilizedaccording to the following protocol. A solution 10 mM of Eu-dragit RL 100 in ethanol 95% was prepared. A solution 10M of FITC in ethanol was added to the solution of the polymer(1:1) and few drops of the cocktail were deposited on the PMMAchannels. After the total evaporation of the solvent, the PMMAsurface was washed with pure distilled water. McIlvaine bufferswith constant ionic strength (1M) were used as solutions for thecharacterization of the pH sensor. Different pH values were ob-tained by mixing citric acid and sodium phosphate dodecahy-drate in appropriate proportions. Potassium chloride was addedin order to obtain the 1 M ionic strength [11].

For the covalent immobilization of the mouse-IgG, thePMMA surface was covered with a solution 10 M of EudragitL 100 in ethanol 95%. After the total evaporation of the solvent,the PMMA surface was washed with pure distilled water andthe carboxylic groups present on the surface of the polymerfilm were activated by means of EDC (2 mM) and NHS (5 mM)in MES buffer 0.1 M at pH 4.5–7.2. After 30 min, the channelwas rinsed with the MES buffer and then with the PBS buffer(137 mM NaCl, 10 mM Na HPO , 2.7 mM KCl, pH 7.4). The2.5 mg/ml solution of mouse-IgG in PBS was let to interact 1 hfor the immobilization and rinsed with PBS. The incubationwith the Cy5-labeled anti-mouse-IgG (1 mg/ml) was performedfor 1 h. The channel was then rinsed with PBS in order toremove the excess labeled anti-mouse-IgG.

III. RESULTS AND DISCUSSION

The analysis of the preferential directions of the emittedanisotropic fluorescence was carried out with the setup de-scribed in Fig. 1, with fluorescein immobilized on the PMMAcover and with a buffered solution having a pH value equal to8 flowing through the channel. Both the fluorescence emittedat 539 nm and the scattered signal at 488 nm (coming fromthe excitation) were measured by means of a collecting fiberpositioned at different angles with respect to the flow direction.The measurements were performed every 5 . The fluorescenceemission is reported in Fig. 3(a), while the efficiency, i.e., theratio of the fluorescence and the scattered signal, is reported

Fig. 4. Fluorescence in the presence of buffer solutions at two different pH.

Fig. 5. Time response of the pH sensor for different pH steps.

in Fig. 3(b). From these results it was possible to optimize thedetected signal by choosing a collection angle of 50 , as shownin Fig. 3(b). It is noteworthy to observe that the collection ofthe fluorescence at this angle implies a fivefold increase in theratio between the fluorescence signal and the scattered light. Onthe basis of these results, the PMMA cap (the waveguide) endswere cut and polished at 60 , so that most of the rays traveledperpendicularly through the ends.

Fig. 4 shows the optical signal collected from the optical fiberand sent to the spectrometer Ocean Optics S2000 in correspon-dence of the flow of two buffer solutions at pH 4.6 and pH 8,

1308 IEEE SENSORS JOURNAL, VOL. 8, NO. 7, JULY 2008

Fig. 6. (a) Longitudinal and (b) transversal cross sections of the improved PMMA chip with the cylindrical lens located on the cover. The cut and polished end-faceat 60 for the better coupling with the fiber can be seen in the longitudinal section (a).

respectively. It is possible to observe both the scattered and dif-fused light coming directly from the source, centered at 488 nm,and the fluorescent light emitted by the fluorophore and centeredat 535 nm. The fluorescence level at pH 8 is comparable withthe excitation signal collected by the GRIN coupled fiber. It isnoteworthy to stress that the adopted geometrical configurationof the optical system allows to collect a high fluorescence signalwith a low background coming from the source. Fig. 5 shows thetime response of the system in correspondence of different pHbuffer solutions flowing through the channel.

In the measurements performed with the Cy5-labeled anti-mouse IgG sandwich assay, the excitation was carried out withthe laser diode emitting at 635 nm, as already specified in theprevious section. An improvement in the excitation process wasachieved with the use of a cylindrical piano-convex lens locatedon the waveguide (Fig. 6), taking care that no index matching isbetween the lens and the cap as to avoid the modification of thewaveguide characteristics. The lens was fixed on the cap simplyby gluing the ends [Fig. 6(a)]. The lens was 15 mm long and2 mm large, and was manufactured by polishing longitudinallya piece of a 2 mm plastic optical fiber. A comparison of the flu-orescence spectra with and without the lens is shown in Fig. 7,in the case of the interaction of Cy5-labeled anti-mouse-IgG(1 mg/ml) with the immobilised layer of mouse-IgG. The col-lected signal is five times greater in the presence of the cylin-drical lens. It is significant to observe that the signal increaseis practically absent, if there is no air gap between the lens andthe chip; or better, a slight decrease in the optical efficiency ofthe system is observed since the lens itself become part of thelight waveguide, giving rise to an increase in the losses of thefluorescence guided through the plastic cap.

IV. CONCLUSION

The proposed system shows promising performances in viewof its utilization in the development of fluorescent-based chem-ical and biochemical sensors. The plastic chip has a cost po-tentially very low, thanks to the possibility of a production bymeans of injection molding. Moreover, the upper part of the flowcell can be chemically functionalized depending on the type ofthe biosensor, and it is easy to be replaced. The particular con-figuration of the system based on the anisotropic behavior of the

Fig. 7. Comparison of the collected fluorescence spectra with and without thecylindrical lens, in the case of the mouse-IgG/Cy5-labeled anti-mouse-IgG in-teraction. The collected fluorescence in the presence of an index matching gelwith the lens between the lens and the chip is also shown.

fluorophore immobilized on the PMMA channel implies a ge-ometrical filtering of the scattered light, offering a substantialcontribution to the decrease of the background in the fluores-cence signal.

In the IgG sandwich assay, all the measurements were carriedout with a very high concentration of labeled IgG, in order to besure that all the available sites for the immunoreaction were oc-cupied, since the purpose of the assay was the evaluation of thecapability of the developed chip to be used as immunochip. Thefull characterization of the immunoassay, with the determina-tion of the limit of detection and sensitivity is in progress.

REFERENCES

[1] T. Vo-Dinh, , T. Vo-Dinh, Ed., “Biochips and microarrays: tools for thenew medicine,” in Biomedical Photonics Handbook. Boca Raton, FL:CRC Press, 2003, ch. 51, pp. 1–29.

[2] J. Homola, H. B. Lu, G. G. Nenninger, J. Dostàlek, and S. S. Yee, “Anovel multichannel surface plasmon resonance biosensor,” Sens. Ac-tuat. B, vol. 76, pp. 403–410, 2001.

[3] G. Gauglitz, A. Brecht, G. Kraus, and W. Nahm, “Chemical and bio-chemical sensors based on interferometry at thin (multi-) layers,” Sens.Actuat. B, vol. 11, pp. 21–27, 1993.

[4] F. Dieterle, G. Belge, C. Betsch, and G. Gauglitz, “Quantification of therefrigerants R22 and R134a in mixtures by means of different polymersand reflectometric interference spectroscopy,” Anal. Bioanal. Chem.,vol. 374, pp. 858–867, 2002.

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[5] F. S. Ligler, C. A. Rowe-Taitt, L. C. Shriver-Lake, K. E. Sapsford, Y.Shubin, and J. P. Golden, “Array biosensor for detection of toxins,”Anal. Bioanal. Chem., vol. 377, pp. 469–477, 2003.

[6] C. Barzen, A. Brecht, and G. Gauglitz, “Optical multiple-analyte im-munosensor for water pollution control,” Biosens. Bioelectron., vol. 17,pp. 289–295, 2002.

[7] J. R. Epstein, I. Biran, and D. R. Walt, “Fluorescence-based nucleicacid detection and microarrays,” Anal. Chim. Acta, vol. 469, pp. 3–36,2002.

[8] G. Proll, J. Tschmelak, and G. Gauglitz, “Fully automated biosensors forwater analysis,” Anal. Bioanal. Chem., vol. 381, no. 1, pp. 61–63, 2005.

[9] H. Stuart and D. Hall, “Enhanced dipole-dipole interaction betweenelementary radiators near a surface,” Phys. Rev. Lett., vol. 80, pp.5663–5666, 1998.

[10] L. Polerecky, J. Hamrle, and B. D. MacCraith, “Theory of the radiationof dipoles within a multilayer system,” Appl. Opt., vol. 39, no. 22, pp.3968–3977, 2000.

[11] T. C. McIlvaine, “A buffer solution for colorimetric comparison,” J.Biol. Chem., vol. 49, pp. 183–186, 1921.

Francesco Baldini received the Degree in physics(magna cum laude) from the University of Florence,Florence, Italy, on February 25, 1986.

Since 1986, he has been with the Optical FiberGroup at the Institute of Electromagnetic Wave, CNR,Florence (now named Institute of Applied Physics).His research activity was devoted to the developmentof optical fiber sensors and to the application ofoptical methods to the restoration of paintings andfrescoes. Now, he is active in the field of opticalfiber sensors/systems for chemical and biochemical

parameters and is the author of more than 100 publications on the subject inInternational Journals, in scientific books, and in International ConferenceProceedings.

Dr. Baldini is Chairman of the ASCOS (Advanced Study Course on OpticalChemical Sensors) Series and since 2005 has been a member of the InternationalAdvisory Board of the Journal Analytical and Bioanalytical Chemistry. He is amember of the Steering Committee of the Europt(r)ode Conference on OpticalChemical Sensors and Biosensors and has been member of the Scientific Com-mittee of many International Conferences.

Adolfo Carloni received the Degree in industrialchemistry in 2003 with a thesis on optical sensorsfrom the University of Bologna, Bologna, Italy,and the Ph.D. degree in chemical sciences fromthe University of Florence, Florence, Italy, in 2008,working at the Institute of Applied Physics of CNR,Florence, on optical biosensors.

He was with the Debye Institute of Utrecht, Hol-land, in 2000 for one year and with the Swiss Fed-eral Institute of Technology of Zurich, Switzerland,in 2004, for six months. He was also with the Cran-

field Health Research Center, U.K., in 2008, for three months. His main researchinterests regards the area of optical biosensors, DNA-protein interactions, mi-croscopy imaging, control deposition of polymers, and more in general indus-trial chemistry and nanotechnology of bioprocesses.

Ambra Giannetti received the Laurea degreein pharmaceutical chemistry and technology andthe Ph.D. degree in medical physiopathology andpharmacology from the University of Pisa, Pisa,Italy, in 2001 and 2005, respectively. She spent herfirst period of the Ph.D. at CNR, Institute of ClinicalPhysiology, Pisa, (January 2002–August 2003), thenat the Oak Ridge National Laboratory, Oak Ridge,TN (September 2003–July 2004).

Presently, she is at the Institute of Applied Physics,Florence, Italy. Her research activity is focused on the

study and development of optical biosensors and biosurfaces including proteinand DNA treatment for preparation of active surfaces.

Andrea Mencaglia received the Degree in physicsfrom the University of Florence, Florence, Italy, in1987.

He was with CNR at the Institute of Researchon Electromagnetic Waves (IROE) since 1987 untilApril 1996, first awarded with fellowships and thenas Researcher. In 1996, he was Research Fellow atthe University of Strathclyde, Glasgow, U.K., for sixmonths within the Human Capital Mobility Program.From January 1997 to 2001, he was responsible forresearch activity at Prodotec. Since January 2002,

he has been a Scientist at the Institute of Applied Physics, CNR. His activityis mainly concentrated in the area of optical fibers sensors and systems andoptoelectronic instrumentation, with applications in the fields of diagnosticsfor food, for the cultural heritage, environmental, industrial, and medical diag-nostics. He is responsible for a contract with a company for the developmentof a system for multiple analysis for clinical diagnostics and collaborates inseveral European and National projects. He is author of about 100 publicationsin Journals and Conference Proceedings and of several patents (U.S., Europe,and Italy).

Giampiero Porro received the Degree in mechanicalengineering from the Politecnico of Milan, Milan,Italy, in 1978.

Since 2003, he has been the Head of InnovationActivities at DATAMED. His track record includesleading teams in the design, manufacture, and com-mercialization of innovative diagnostic and therapeu-tical equipment for Diabetes treatment at ESAOTE-BIOMEDICA, the development of online blood mon-itoring equipment at SORIN BIOMEDICA, and themanufacture and launch of disposable medical de-

vices for Dialysis and Artificial Nutrition at FRESENIUS MEDICAL CAREGmbH.

Lorena Tedeschi received the Laurea degree inpharmaceutical chemistry and technology and thePh.D. degree in molecular biotechnologies from theUniversity of Pisa, Pisa, Italy, in 1999 and 2003,respectively.

Since 1999, she has been involved in researchactivities on biosensors and biosurfaces at Centro“E. Piaggio,” University of Pisa and CNR Institute ofClinical Physiology, Pisa. Since 2003, she has beenworking at CNR Institute of Clinical Physiology, asa Postdoc Research Assistant (until 2006) and as a

Researcher (from 2007 to now). Her main research interest are in the area ofDNA-based and protein-based biosensors, specifically about design, synthesisand purification of bioreceptors and their labeling and immobilization onvarious kinds of transducers.

Cosimo Trono received the Degree in physics andthe Ph.D. degree from the University of Florence,Florence, Italy, in 1998 and 2003, respectively.

Since 1998, he has been with the Fiber OpticsGroup at the Institute of Electromagnetic Waveof CNR, Florence, Italy (now named Institute ofApplied Physics “Nello Carrara”), with temporarycontracts. His research activity was devoted to thedesign and development of optical sensors and tothe study of novel optical methods for sensing.Recently, he has become active in the field of optical

sensors for chemical and biochemical parameters. He is holder of internationalpatents and author of more than 40 publications in International Journals andin International Conference Proceedings.

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