Development of a PDMS microfluidic system.

AA POLYDIMETHYSILOXANE(PDMS)-based microfluidic system withpneumatic micropumps and micro-mixers has been developed for high-accuracy insulin detection of clinicalplasma samples. The detection methodis based on double-antibody sand-wich immunoassay coupled withluminol-hydrogen peroxide (H2O2)chemiluminescence.

Diabetes mellitus is a group ofdiverse metabolic disorders caused bydeficiency or diminished effectivenessof endogenous insulin [1]. It is thethird leading cause of disease-relateddeath around the world after cancerand cardiovascular disease. At present,the blood glucose test is the most popu-lar clinical diagnostic tool for diabe-tes, but irregular blood glucose level,

unfortunately, is not a good early indi-cator for diabetes diagnosis. Previousstudies have shown that insulin, con-necting peptide, and interleukin-6 arebetter suited as early diagnostic im-mune indicators for the onset of diabe-tes [2]. Insulin, a hormone producedby the pancreas for converting glucoseto energy, is a common clinical indica-tor. Its abnormity can be detected as an

Digital Object Identifier 10.1109/MNANO.2011.941954

Date of publication: 24 August 2011

ZHIKUN ZHAN, ZAILI DONG, AND STEVE TUNG

© PHOTODISC

12 | IEEE NANOTECHNOLOGY MAGAZINE | SEPTEMBER 2011 1932-4510/11/$26.00©2011IEEE

effective auspice to diagnose diabetes atan early stage, which is helpful for dia-betes prevention. The traditional testmethods for insulin and other indica-tors in clinical diagnosis, which includeenzyme-linked immunosorbent assay(ELISA), radioimmunoassay, and vari-ous chemiluminescence-based techni-ques, are usually based on microplatesand large automatic biochemistry ana-lyzers. These methods, while effective,are predominantly laboratory basedand require trained technicians. Thismakes them unsuitable for field testing,which is becoming increasingly im-portant for diabetes screening in devel-oping countries.

Microfluidics, with advantages suchas low reagent consumption, short anal-ysis time, high reliability and sensitivity,and multiprocess integration [3], [4], isa rapidly developing field in biomedicaltesting [5]. In microfluidic systems, theconventional biochemical processes, suchas sample pretreatment, sample/reagenttransport, mixing, reaction, separation,detection, and product collection, can becarried out automatically on a single chip[6]. Additionally, microfluidic systemshave become the most promising tool forhandling costly and difficult-to-obtainsamples and reagents [7]. More impor-tantly, the portability characteristic ofthese systems that arises from theircompact form is a key factor for point-of-care applications [6] and offers apractical solution to diabetes screeningin a rural environment.

Recently, several advancements inmeasuring insulin using microfluidicdevices coupled with novel detectiontechniques were demonstrated. A car-bon nanotube/dihydropyran compositesensor in a microfluidic device has beenreported to have a detection limit of1 lM [8]. A SlipChip-based approachwas demonstrated to perform bead-based heterogeneous immunoassays withmultiple nanoliter-volume samples. Aninsulin immunoenzymatic assay showeda detection limit of about 13 pM [9].Another approach used a microfluidiclocal-surface plasmon resonance chipoperated with a simple collinear opticalsystem to interrogate specific insulin andantiinsulin antibody reaction in real time

and demonstrated a detection limit of100 ng/mL insulin [10]. Furthermore,by conjugating with a continuous-flowimmunosensor, the detection limit ofthe surface plasmon resonance (SPR)method can be improved to 1 ng/mL(ppb) with a response time of less than5 min [11]. However, these methodsstill require labor-intensive processing,large equipment, and trained experts toprovide accurate insulin measurements.

In the present study, an integratedPDMS-based microfluidic system coupledwith magnetic bead-based sandwichELISA has been developed to measureinsulin concentrations. Superparamagneticbeads coated with insulin antibodies areused to capture the target insulin mole-cules. The integrated microfluidic systemthat is composed of two pneumatic micro-pumps and a micromixer automates theimmunoreaction process. A chemilumi-nescent detector is then used to quantifythe concentration of the insulin capturedby the immunobeads in the micromixer.Using this approach, the developed micro-fluidic system achieves a low detectionlimit and a short reaction time.

MATERIALS AND METHODSFigure 1 shows a schematic illustrationof the experimental procedure for in-sulin detection using heterogeneouschemiluminescent immunoassays, whichare typically performed manually in bio-medical laboratories. In this procedure,carboxyl-modified magnetic microsphereswith a mean diameter of approximately5 lm are used as solid-phase supports tocovalently conjunct primary insulin anti-bodies in centrifuge tubes [Figure 1(a)].The immunocompetent antibodies con-jugated on the bead surface allow thebeads to capture the target insulin (anti-gen). Taking advantage of the specificinteraction between the immune beadsand insulin, the target insulin molecules

are then recognized and form a halfsandwich structure in the micromixerchamber with the microbeads. In thepresence of an external magnet, thenonreactive insulin molecules are washedaway [Figure 1(b) and (c)]. Then theinsulin antibodies labeled with horserad-ish peroxidase (HRP) are added to formcomplete sandwich complexes [Figure 1(d)and (e)].

After removing the nonreactivematerials, luminal and H2O2 with theenhancer p-iodophenol are injected.Under the condition of alkalescence andcatalyzed by the HRP of the sandwichcomplexes, the injected luminol israpidly oxidized by H2O2 and emitslight at a wavelength of approximately425 nm with an intensity proportionalto the quantity of HRP [Figure 1(f)].Since the insulin and its secondary anti-body are a one-to-one conjugation, thequantity of HRP connected to thesecondary Ab is a direct indication of thequantity of insulin. The emitted light isquantified by a luminometer. All insulin-related bioreagents are obtained fromShanghai E. Star BioTechnology Co.

The immune-related processes are car-ried out semiautomatically in the micro-fluidic system. Traditionally, the immunemagnetic beads and biosamples are firstincubated for 60 min to recognize the tar-get insulin. After incubation, the targetinsulin adhered onto the magnetic beadsis captured using an external magnet.Then, a wash buffer [phosphate bufferedsaline Tween-20 (PBST)] is added towash away the nonreactive materials. TheHRP-labeled insulin antibody is thenmanually added and allowed to interactwith the target insulin for 30 min. Theunbound, HRP-labeled insulin antibodyis then washed away. Finally, luminol,H2O2, and the enhancer (p-iodophenol)are added for chemiluminescence detec-tion using a luminometer.

Diabetes mellitus is a group of diverse metabolicdisorders caused by deficiency or diminished

effectiveness of endogenous insulin.

SEPTEMBER 2011 | IEEE NANOTECHNOLOGY MAGAZINE | 13

COOHCOOH

MESEDAC

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COOH Carboxyl-ModifiedMagnetic Microbead

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Insulin Antibody

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FIGURE 1 Experimental procedure for insulin detection using heterogeneous chemiluminescent immunoassays.

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MICROFLUIDIC SYSTEMThe integrated microfluidic system iscomposed of two PDMS-based pneu-matic micropumps and a micromixer.Tygon tubes connect the outlets of themicropumps to the inlets of the micro-mixer, as shown in Figure 2.

The PDMS-based pneumatic micro-pump and micromixer have similarstructures (Figure 3) and working prin-ciples. Both designs consist of two thinPDMS layers. The top layer containseither a microchannel (micropump) ora mixing chamber (micromixer) witheither an inlet and an outlet or twoy-shaped inlets and an outlet. The heightof the microchannel is 100 lm while thediameter and depth of the mixing cham-ber are 2 mm and 100 lm, respectively.The micropump and micromixer areoperated based on pneumatic peristalticprinciples. Their bottom PDMS layerconsists of either three (micropump) orsix (micromixer) membrane-enclosedair chambers. In the micropump, thethree serially connected air chambershave different volumes and generatefluid movement in the microchannelthrough a sequential diaphragm deflec-tion [12]. In the micromixer, the six airchambers have identical volumes. Mix-ing is achieved by pneumatically deflect-ing the six 100 lm-thick diaphragms ofthe air chambers. The chambers areconnected in series to each otherthrough serpentine microchannels. Aminiature air compressor is used to sup-ply the required compressed air and is

controlled by an electromagnetic valve(EMV). Details of the fabrication processflow for the micropump and micromixercan be found in [13].

EXPERIMENTAL RESULTSAND DISCUSSIONS

EXPERIMENTAL SETUP

Figure 4 demonstrates the experimentalsetup of the insulin detection scheme.The setup consists of a mini air compres-sor and a custom-designed circuit thatcontrols the opening and closing ofEMV to control the air flow into themicropump and micromixer. A pressuresensor is utilized to monitor the air pres-sure. The chemiluminescence signal fromthe micromixer is detected by a photom-eter (RFL-1A).

CHARACTERIZATIONS OF

THE PNEUMATIC MICROPUMP

AND MICROMIXER

The micropump and micromixer areextensively tested before insulin detec-tion to determine their operatingparameters. The effects of the appliedair pressure and the EMV driving fre-quency on the resultant flow rate of themicropump are explored using ultrapure water. As shown in Figure 5, thegeneral trend of the flow rate under aconstant pneumatic pressure is ascendingwith the increasing driving frequency.

(a)

(b)

FIGURE 2 (a) PDMS micropump and(b) micromixer.

Microfluidics is a rapidly developingfield in biomedical testing.

Mixing Chamberor Microchannel

Fluid In Fluid Out Thin-MembraneAir Chambers

Glass Slide

Glass Slide

Air In Air Out

PDMS-TopLayer

PDMS-BottomLayer

FIGURE 3 Cross-sectional view of the PDMS micromixer/micropump.

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However, the maximum achievable flowrate is limited by the speed of the fillingand emptying of the air chambers. Whenthe EMV frequency is too high, thePDMS diaphragm cannot completelydeflect and recover back to its originalstate. This causes the flow rate to fall afteran optimal frequency is reached.

The micromixer’s performanceis also evaluated under differentapplied air pressures and EMVdriving frequencies. The peakchemiluminescent light intensitywhen the reagents flow throughthe mixing chamber at a constantflow rate of 25 lL/min is used asa measure of the mixing efficiency.The chemiluminescent reagent con-centration used is 4:8 3 10�3 Mfor luminol and 7:5 3 10�3 M forH2O2. The result indicates thatat 5 and 10 lbf/in2, the peakchemiluminescent intensity increaseswith the increasing frequency up to10 Hz. At 15 lbf/in2, the lightintensity decreases with the increasein frequency. On the basis of this

result, it is determined that the optimalworking parameters of the micromixerwill be 10 lbf/in2 and 5 Hz.

INSULIN MEASUREMENT

The serum samples used in the presentstudy were obtained from diabeticpatients at the Chinese Medical

University Hospital, Shenyang, China.Since the insulin concentration is almostthe same as our detection limit of10�10 M, the serum sample is pretreatedby adding nonclinical insulin to makehigher concentration solutions. Thenonclinical insulin is purchased fromShanghai E. Star BioTechnology Co.

Nonclinical insulin is dilutedby phosphate buffered saline(PBS) (pH 7.4) to be 9.5 3

10�7 M, 4.75 3 10�8 M, and4.75 3 10�9 M. The insulinoriginal concentrations in clini-cal postprandial serums of threedifferent patients are measuredby a biochemical analyzer. They are68.94, 42.25, and 37.53 mIU/L,respectively, but become 4.75 3

10�7 M, 2.39 3 10�8, and 5.0 3

10�9 M after adding nonclinicalinsulin.

The insulin measurement pro-cess is divided into two proce-dures. The immunoreactions andthe related washing steps arecarried out in the integrated

Fluid 1 Fluid 1

Fluid 2

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Valve Valve

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FIGURE 4 Integrated microfluidic system with two pneumatic PDMS micropumps and a micromixer.

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FIGURE 5 Flow rates of the pneumatic micropump under differentoperating parameters.

16 | IEEE NANOTECHNOLOGY MAGAZINE | SEPTEMBER 2011

microfluidic system shown inFigure 2. The chemiluminescentreaction is accomplished in a sepa-rate micromixer with the help of asyringe pump for reagent injection.For the microfluidic system, thebiochemical reagents and washbuffers are delivered into the mix-ing chamber by two micropumpsoperated at an almost identicalpumping rate of approximately11 lL/min. The mixer is operatedat a driving frequency of 5 Hz andan applied air pressure of 10 lbf/in2.

The immunoreaction processbegins with the transport of conju-gated magnetic beads produced from theprocess described in Figure 1(a) andthe insulin solution into the mixingchamber to form half-sandwich struc-tures [Figure 1(b) and (c)]. Then, thesestructures and the HRP-labeled insulinantibodies are brought together to formthe sandwich complex [Figure 1(d) and(e)] for the subsequent chemilumines-cent reaction. Washing procedures arecarried out following each immune reac-tion with the help of an external perma-nent magnet.

The micromixer used for the chemi-luminescence detection is placed in thedark box of the photometer to reducenoise and improve sensitivity. A double-channel syringe pump is used to injectreagents into the mixing chamber via aY-shaped microchannel. The solutioncontaining the HRP-labeled insulin sand-wich complex, hydrogen oxide (7.5 3

10�3 M), and the enhancer p-iodophenol(3 mg/mL) is injected by one syringewhile a second syringe is used to deliverthe luminol solution (7.8 3 10�3 M).The two solutions are allowed to blendwell and react [Figure 1(f)].

The three samples with differentinsulin concentrations are examinedfour times within a single day andrepeated three times every two days.Figure 6 demonstrates the average sensi-tivity curve. Overall, the result indicatesthat the detection limit is 10�11 M witha linear range of 5 3 10�10 M–6.4 3

10�9 M. The repeatability test resultsindicate an average coefficient of variation

(CV) value of 14.95% for samples in thesame batch and 15.70% for differentbatches. This implies that the insulin de-tection method investigated in this articleis more consistent for samples within thesame batch. The detection time of themicrofluidic system is about 10 min.

CONCLUSIONSThe current study demonstrates theapplication of a PDMS microfluidic sys-tem for insulin detection of clinicalplasma samples based on heterogene-ous chemiluminescent immunoassays.The detection method utilizes twopneumatic micropumps operated at aflow rate of about 10 lL/min and amicromixer operated at a frequency of5 Hz. The detection limit of the PDMSmicrofluidic system is 10�11 M with alinear range of 5 3 10�10 M – 6.4 3

10�9 M. The detection time is about 10min, which is more than ten times fasterthan that of the conventional methodsfor insulin detection.

ACKNOWLEDGMENTThis project is funded by the ChineseState Key Laboratory of Robotics(grants RLZ200808 and RLO200812)and the National High TechnologyResearch and Development Program ofChina (grant 2009AA04Z313).

ABOUT THE AUTHORSZhikun Zhan (Zhanzhikun2010@gmail.com) was a Ph.D. student at the StateKey Laboratory of Robotics, Shenyang

Institute of Automation, ChineseAcademy of Science, when theresearch work described in thearticle was carried out. Currently,she is a lecturer at the Automa-tion Department, Institute ofElectrical Engineering, YanshanUniversity, China.

Zaili Dong (dzl@sia.cn) is aprofessor at the State Key Labo-ratory of Robotics, Shenyang In-stitute of Automation, ChineseAcademy of Science.

Steve Tung (chstung@uark.edu) is an associate professor at theDepartment of Mechanical Engi-

neering at the University of Arkansas.

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[8] R. M. Snider, M. Ciobanu, A. E. Rue, andD. E. Cliffel, ‘‘A multiwalled carbon nanotube/dihydropyran composite film electrode for insulindetection in a microphysiometer chamber,’’ Anal.Chim. Acta, vol. 609, no. 1, pp. 44–52, 2008.

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[10] H. M. Hiep, T. Nakayama, M. Saito, S.Yamamura, Y. Takamura, and E. Tamiya, ‘‘Amicrofluidic chip based on localized surfaceplasmon resonance for real-time monitoring ofantigen-antibody reactions,’’ Jpn. J. Appl. Phys.,vol. 47, no. 2, pp. 1337–1341, 2008.

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FIGURE 6 Relationship between insulin concentration and thecorresponding chemiluminescence signal relative luminescenceunits (RLUs).

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