Lab on a Chip

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A micro-cantilever sensor chip based on contact angle analysis for a label-free troponin I immunoassay3
Cite this: Lab Chip, 2013, 13, 834

Tsung-I Yin,*a Yunpeng Zhao,a Josef Horak,a Huseyin Bakirci,a Hsin-Hao Liao,b HannHuei Tsai,b Ying-Zong Juangb and Gerald Urbana
Cantilever sensors have been extensively explored as a promising technique for real-time and label-free analyses in biological systems. A major sensing principle utilized by state-of-the-art cantilever sensors is based on analyte-induced surface stress changes, which result in static bending of a cantilever. The sensor performance, however, suffers from the intrinsically small change in surface stress induced by analytes, especially for molecular recognition such as antigenantibody binding. Through the contact angle change on a tailored solid surface, it is possible to convert a tiny surface stress into a capillary forcea much larger physical quantity needed for a practical sensor application. In this work, a micro-cantilever sensor based on contact angle analysis (CAMCS) was proposed to effectively enhance the sensitivity of a sensor in proportion to the square of the length to thickness ratio of the cantilever structure. CAMCS chips were fabricated using a standard complementary-metal-oxide-semiconductor (CMOS) process to demonstrate a 1250-fold enhancement in the sensitivity of surface stress to bioanalyte adsorption using a piezoresistive
Received 7th July 2012, Accepted 30th November 2012 DOI: 10.1039/c2lc40767a www.rsc.org/loc

sensing method. A real-time and label-free troponin I (cTnI) immunoassay, which is now widely used in clinics and considered a gold standard for the early diagnosis and prognosis of cardiovascular disease, was performed to demonstrate cTnI detection levels as low as 1 pg mL21. The short detection time of this assay was within several minutes, which matches the detection time of commercially available instruments that are based on fluorescence-labeling techniques.

Introduction
Cardiovascular disease (CVD) is the single greatest cause of adult mortality in the western world and imposes a huge burden on healthcare services.1 The successful early diagnosis and prognosis of CVD relies on the identification of cardiac biomarkers that can be used to detect cardiac distress and that add value to current risk stratification criteria.2 Troponin I (cTnI), a very sensitive and reliable biomarker of damaged cardiac tissue, is now widely used in clinics and is considered a gold standard.3 There is now a clear movement towards ultrasensitive detection (demonstrating sensitivity ,0.1 ng mL21) of those biomarkers to meet the required 99th percentile reference value for cTnI with the aim of point-ofcare (POC) diagnostics.47 Some commercially available instruments, such as PATHFAST1 by Mitsubishi Chemical Europe GmbH and Stratus1 CS STAT by SIEMENS Health Diagnostics Inc., have been developed for POC tests for cTnI,
Dept. for Microsystems Engineering (IMTEK), University of Freiburg, 79110 Freiburg, Germany. E-mail: tsung-i.yin@imtek.uni-freiburg.de; Tel: +49-(0)761/ 203-7269 b National Chip Implementation Center, National Applied Research Laboratories, 300 Hsinchu, Taiwan 3 Electronic supplementary information (ESI) available. See DOI: 10.1039/ c2lc40767a
a

with analytical sensitivity in the range of sub-ng mL21.2 The operation of those devices, however, is limited by the requirement for a label for conventional enzyme-linked immunosorbent assay (ELISA), which adds significant costs and time for development.8 Label-free technologies for quantitative multiparameter biological analysis provide another option for immunoassays.8 In recent years, cantilever-based sensors have been extensively explored as a promising technique for label-free analyses, such as hybridization of deoxyribonucleic acid (DNA), antibodyantigen binding, small ion detection, toxic gas detection, and intermolecular interaction of a selfassembling monolayer.911 One of the major sensing principles utilized by these sensors is the so-called surface stress mode, in which a change in surface stress induced by the immobilized analytes on cantilever surfaces is converted into a static deflection of the cantilevers for detection.10,12 The sensor performance, however, suffers from the intrinsically small change in surface stress induced by analytes in biological systems, such as a typical stress of 110 mN m21 for antigenantibody binding.10 The low signal-to-noise ratio (SNR) became the main obstacle in commercializing those cantilever sensors for detecting low concentration samples.13 Different approaches such as utilizing materials with a lower elastic modulus14,15 and modifying cantilever geometries1618

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Lab on a Chip have been proposed in an attempt to increase the sensitivity to surface stress, thus increasing the SNR of the sensor. A 20-fold enhancement in the sensitivity to surface stress has been recently demonstrated in a membrane-type cantilever sensor.18 The working environment of the sensor, however, is the gas phase with a surface stress loading of approximately 3 N m21,18 which is far larger than the 110 mN m21 that is the aim of detection in liquid phases. For label-free immunoassays, an effective approach that can improve the force sensitivity of cantilever sensors is mandatory. Under ambient conditions, the capillary forces formed by sessile liquid micro-drops on a cantilever surface or by the liquid meniscus in tipsurface interactions between an atomic force microscope tip and a substrate surface have been shown to dominate other surface forces.1921 The magnitude of capillary forces is correlated with surface properties through a change in the contact angle at solidliquid interfaces.22 This physical phenomenon implies that a tiny intermolecular force can be converted into a capillary forcea much larger physical quantitythrough a change in the contact angle. Moreover, the contact angle itself is very sensitive to angstrom-scale details of the interfacial surface and has proven to be invaluable in probing the character of solidliquid interfaces involving organic solids,23 e.g., the change in the solidliquid interfacial tension induced by DNA hybridization and tumor angiogenic ligandreceptor protein interactions.24,25 By utilizing such a sensing and force amplification mechanism, it is possible to produce a cantilever sensor with ultrasensitive performance.26 Based on our preliminary proof of concept study,26 a m _icroc _antilever s _ensor based on c _ontact a _ngle analysis,

Paper named CAMCS, was further developed for a real-time and label-free troponin I immunoassay. A mechanics model was proposed to describe the sensitivity of a piezoresistive multilayer cantilever under capillary force loading in accordance with the change in contact angle of a liquid meniscus generated by microfluidics. A CAMCS chip, implemented using a standard complementary-metal-oxide-semiconductor (CMOS) process, with an integrated microfluidics system was proposed for liquid phase measurements. An experiment with bovine serum albumin (BSA) adsorption was performed to characterize the CAMCS chips. Finally, a real-time and labelfree troponin I immunoassay was performed to demonstrate the merit of the CAMCS design.

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Materials and methods

CAMCS design Fig. 1 (a) schematically depicts the design of a CAMCS. Two isolated cantilevers with length L, width W, and thickness t were placed within an integrated microfluidic channel. To generate a liquid meniscus between the edges of a cantilever and its surrounding boundary, a micro-slit with gap width gx was produced during the fabrication process.27 The step height between the top surface of the cantilevers and the surrounding boundary is gz. Under normal microfluidic working pressures and channel sizes in the range of micrometers, the liquid sample flow will be restricted to the top surface of the cantilever because of the far larger flow resistance and surface tension of the liquid. Based on the wetting morphologies of liquid in grooves with a rectangular cross section,28 the contact line of the meniscus will pin along

Fig. 1 (a) Schematic illustration of a CAMCS design. A pair of cantilevers with a surrounding micro-slit (gap width gx and step height gz) is placed under a microfluidic channel to generate a liquid meniscus between the edges of a cantilever and its surrounding boundary through the injection of a liquid sample. The contact line of the meniscus remained at the periphery of the cantilever and the surrounding boundary, forming a changeable contact angle h.This contact angle is determined by a force balance between the solidliquid interfacial tension csl and the horizontal component of the surface tension of liquid clg cos h. A change in solidliquid interfacial tension, Dcsl , induced by adsorbed analytes on the cantilever surface yields a change in the contact angle Dh and thus an accompanying change in capillary pressure DP and surface tension Dc. (b) A comparison of the normalized sensitivity of a piezoresistive cantilever based on capillary forces and surface stress measurement against the length to thickness ratio (L/t) of the cantilever structure.

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Paper the edges of the cantilever and the surrounding boundary, forming a changeable contact angle, h. This contact angle is determined by the force balance between the solidliquid interfacial tension csl and the horizontal component of the surface tension of liquid clg cos h. The meniscus curves either toward the liquid or away from the liquid to minimize the free energies28 and generate a Laplace pressure on the cantilever as   1 1 P~clg z (1) r1 r2 where r1 and r2 are the principal radii of curvature along the orthogonal axes of the meniscus. According to the geometry of the proposed CAMCS design, r1 = , and r2 can be expressed by r2(h) = 21 h /(2sin( h2 h0)), where h0 is equal to tan (gz/gx), and h is equal to p 2 2 gz zgx . In addition to the loading of the Laplace pressure on the cantilevers, the normal component of the surface tension of the meniscus, clg sin h, poses another uniformly distributed force along the edge of the cantilevers. The total capillary forces, including the effect of the Laplace pressure and surface tension, are compensated by the elastic response of the cantilevers. Capillary force change induced by a change in solidliquid interfacial tension Altering the surface properties of the cantilever, e.g., through a physisorption of proteins, will change the contact angle of the meniscus, Dh, in accordance with a change in the solidliquid interfacial tension Dcsl (using the Shuttleworth equation, which assumes small surface strains,29 the magnitude of Dcsl has been shown to be approximately equivalent to the magnitude of surface stress change, Dss). Assuming that the change in surface tension of the liquid sample is negligible, Dcsl can be expressed as Dcsl ^clg cos h{ cos h, where h9 = h + Dh. Under a change in the contact angle of the meniscus, the accompanied change in the Laplace pressure is ! 1 1 DP~clg (2) 0 { r2 (h ) r2 (h) In addition, the change in the normal component of the surface tension induced by Dh is Dc~clg sin (h){ sin h (3)

Lab on a Chip   ! DPW z2Dc L{x2 zDcW L{x 2

ex ~ P

z i Ei Ii

(4)

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where z is the displacement from the neutral axis of the multilayer structure. Ei and Ii are the Youngs modulus and the moment of inertia of each structure layer, respectively. The sensitivity of the piezoresistive cantilever, S, can be represented as the average values of the relative resistance change of the piezoresistor (DR/R)avg, which is equal to Gex dx= dx, where is the length of the piezoresistor, and
0 0

G is the gauge factor. A detailed derivation of the mechanics model of a multilayer cantilever for capillary forces detection is described in the ESI.3 The merits of CAMCS design Assuming that the material properties in each layer of the cantilever in eqn (4) are the same and that z = 1/2t, the force sensitivity of the simplified cantilever can be derived as S^3G=2E DPz2Dc=W zDc=LL=t2 . In contrast, conventional cantilever sensors designed to measure a surface stress change, Dss, have sensitivity S^3G1{nDss =E 1=t according to the Stoney equation.13 The normalized S from these two mechanisms can be plotted against the ratio of L/t as shown in Fig. 1 (b). Taking advantage of the capillary forces, the sensitivity of the CAMCS design can be significantly amplified by several orders of magnitude with (L/t)2. The reason for the dramatic amplification in sensitivity is that the capillary forces apply normal force loading rather than an in-plane force on the cantilever to facilitate higher cantilever deflection.17 Meanwhile, the magnitude of capillary force loading is proportional to the surface area of a cantilever, whereas the magnitude of surface stress loading is geometry independent. With this strategy, there is a strong possibility for increasing the sensitivity by several orders of magnitude with the CAMCS design. Fabrication of CAMCS chips CAMCS chips were implemented using a standard 0.35 mm 2P4M (2 poly/4 metal) CMOS process from TaiwanSemiconductor-Manufacturing-Company (TSMC)30 with postCMOS micromachining to release the cantilever structures and make polydimethylsiloxane (PDMS) microfluidic channels. As shown in Fig. 2 (a), a multilayer structure was sequentially deposited based on the 2P4M CMOS process, where n+ doped polysilicon (poly 2) at a concentration of (15) 6 1020 atom cm23 was used for the piezoresistive layer. Metal layers 14 and tetraethoxysilane (TEOS) inter-metal dielectric layers were stacked as structural layers and as a metal line connection. The trench was formed by anisotropic etching of the TEOS using a metal layer as a hard mask (Fig. 2 (b)). A Ti/Au layer (30/300 nm) was deposited on the cantilever as the molecular sensing layer and protected using a thicker coating of photoresist. Isotropic dry etching of the silicon substrate was then used to release the cantilever structure (Fig. 2 (c)). Finally, a microfluidic channel cover fabricated with a PDMS micromolding process was adhered to the cantilever sensor chip by a stamp-and-stack bonding technique (Fig. 2 (d)).31

Sensitivity of CAMCS by piezoresistive sensing The change in the capillary forces on the cantilevers can be detected using various methods.9,11 One of the most promising methods uses lever-integrated piezoresistive sensing, which does not require bulky and complex instrumentation and can be used in any opaque liquid. The cantilevers in the CAMCS are composed of a top molecular sensing layer and a u-shaped piezoresistor embedded within electrically insulated layers for force detection.16 Therefore, the mechanical strain along the length of cantilever in the piezoresistor is given by

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Lab on a Chip

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Fig. 2 Fabrication of CAMCS chips using a standard 0.35 mm 2P4M (2 poly/4 metal) CMOS process with post-CMOS micromachining. (a) Deposition of metal layers 1 4 and tetraethoxysilane (TEOS) inter-metal dielectric layers as structural layers as well as a metal line connection. (b) Anisotropic etching of TEOS to form the trench. (c) Deposition of a Ti/Au layer as a molecular sensing layer on top of the cantilever and isotropic dry etching of the silicon substrate to release the cantilever structure (d) Adhesion of a PDMS microfluidic channel cover on top of the cantilever sensor chip using a stamp-and-stack bonding technique.

Fig. 3 (a) shows a photograph of a fabricated CAMCS chip with a magnified top view and a scanning electron microscope (SEM) image of a pair of cantilevers. Because of the intrinsic film stress in the CMOS process, the released cantilevers bent upward slightly, which caused a deviation in the step height gz at the end of the cantilever tip. To minimize the effect of residual stress, the dimension of a single cantilever was chosen to be 50 mm in length, and 30 mm in width. With a thickness of approximately 2 mm, the length to thickness ratio L/t is approximately 25. The gap width gx of the micro-slit was chosen to be 10 mm to ensure that the cantilever structure could be fully released with the isotropic dry etching. The step height gz is approximately 3.75 mm with the CMOS process. The piezoresistor embedded within the cantilevers is 40 mm in length and 3 mm in width with a measured resistance of approximately 1.84 kV. The size of the microfluidic channel is 200 mm or 400 mm wide and 100 mm high. Bridge circuit design in CAMCS chips In the CAMCS chips, two pairs of fully symmetric cantilevers located in two isolated microfluidic channels were arranged as a Wheatstone bridge circuit for signal read-out (Fig. 3 (b)). The cantilevers denoted as R1 and R3 within the sensing MC channel are used to detect biochemical analytes, whereas the others denoted as R2 and R4 within the reference (Ref) channel are used for reference. The initial values of R14 were designed to be equal. The signal output of the circuit is therefore given by the relation Vout =Vbias ~1=2DR=Ravg ^1=4G ex~0 , where Vbias is the bias voltage of the bridge circuit, and the estimated gauge factor G is approximately 15 according to the doping concentration during the CMOS process.32 Compared with the former sensor design for liquid phase measurement using piezoresistive cantilevers,33 the proposed design of fully symmetric cantilevers with isolated microfluidic channels can effectively minimize the signal offset caused by self-heating in cantilevers

with different layer compositions while also reducing the mechanical noise from the measurement environment. Experimental setup Before each experiment, the CAMCS chips were sequentially cleansed using acetone, isopropanol, and deionized (DI) water to remove the photoresist layer that was coated on the cantilever surface in the post-CMOS process. The quality of the Au layer after the cleaning process has been examined beforehand.30 The CAMCS chips were used only for single experiment in the present work and the cantilever sensors were not reused for a new measurement. During the sensor operation, Vbias was provided by a Keithley 2601A DC power supply (Keithley Instrument Inc., Cleveland, Ohio), and Vout was extracted using a Keithley 3706 multimeter (Keithley Instrument Inc., Cleveland, Ohio) integrated with a LabVIEW program (National Instruments Germany GmbH) for multichannel signal readout. To minimize the effect of self-heating induced by the piezoresistor embedded inside the cantilevers, Vbias was set up to 0.1 V. A handheld micropipette was used to inject liquid samples into the microfluidic channels without using any pumping system. Because of the size of the microfluidic channel, a sample volume of less than 1 mL was sufficient to fill the entire channel. BSA and DTSSP-BSA adsorption on gold surface Bovine serum albumin (BSA) was purchased from SigmaAldrich. 3,39-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) was purchased from Thermo-scientific (Pierce Biotechnology, Rockford, IL). For the adsorption of BSA on the gold surface of a cantilever, phosphate buffered saline (PBS; 0.1 M, pH = 7.4) was injected into the microfluidic channel first to rinse the cantilever surface and stabilize the signal. Next, 1 mg mL21 of BSA in PBS was injected for BSA adsorption. The signal output during the adsorption process was monitored in real time. To immobilize BSA on DTSSP, 2 mM of DTSSP in citrate buffer (5 mM, pH = 5) was injected to form a self-assembled monolayer

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Lab on a Chip For the label-free immunoassay, the same protocol for forming a SAM of DTSSP on a gold surface of a cantilever was performed over 1 h at room temperature (RT). After washing with citrate buffer to remove the excess DTSSP in solution, a mixing solution of 10 mg mL21 of MAb (19c7) and 10 mg mL21 of MAb (16A11) in PBS was immediately injected into the sensing MC channel over 1 h at RT to immobilize the MAbs on the SAM of DTSSP via the sulfo-NHS group. After washing with PBS to remove the excess MAbs in the solution, a blocking solution composed of 1% BSA and 0.5% casein in PBS was injected into both the sensing and Ref MC channels over 1 h at RT to block additional adsorption sites on the cantilever surface. After the PBS washing, the CAMCS chips were connected to the measuring instrument to obtain a stable base line of the signal. Different concentrations from 1 pg mL21 to 50 ng mL21 of the ITC complex in PBS were then injected into the sensing MC channel for antigen-binding detection.

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Results and discussion

Characterization of CAMCS chips To characterize the embedded piezoresistor in the cantilevers, a thermo-bending test was performed that utilized a bridge circuit with fixed values of R2 and R4. With a bias voltage Vbias = 0.1 V, thermal stress was induced from the self-heating of the piezoresistor. The cantilevers were bent down because of the higher thermal expansion coefficient of the metal layer in the cantilevers. The induced compressive strain in the piezoresistor corresponded to an increase in the signal output DVout of approximately 1.5 6 1022 mV, which was measured after the signal reached an equilibrium state as shown in Fig. 4 (a). The effect of capillary forces on the CAMCS chips was demonstrated by generating a PBS meniscus within the microfluidic channels. The injection of PBS induced a tiny increase in the signal in A and then a prominent decrease in the signal in B until equilibrium was reached as shown in Fig. 4 (a). The signal change indicated that the PBS meniscus generated a lifting capillary force to bend the cantilevers up. Based on the magnitude of DVout, the contact angle of the meniscus h could be estimated to be 18u using eqn (1) and (4), which is slightly smaller than h0 (y20.5u). The equivalent capillary force generated by the convex meniscus was composed of 5.0 mN from surface tension and 20.9 mN from Laplace pressure. According to the signal curve, the contact angle of the meniscus was approaching equilibrium after 200 s, which is consistent with the spreading kinetics of droplets of PBS on gold surfaces.24 The weight of liquid on top of the cantilevers was estimated to be 1.5 pN, which is negligible compared with the magnitude of the capillary force. The initial increase of the signal in A and thus the smaller h and larger curvature of the meniscus can be explained by the increasing liquid volume during the PBS injection. Signal induced by the adsorption of BSA Fig. 4 (b) shows the signal output of the CAMCS chips in two cases of BSA adsorption: one is the physisorption of BSA onto

Fig. 3 (a) Photograph of a fabricated CAMCS chip with a magnified top view and an SEM image of a pair of cantilevers. The dimensions of a single cantilever are 50 mm in length, 30 mm in width, and thickness in 2 mm. The gap width gx of the micro-slit is 10 mm, and the step height gz is approximately 3.75 mm considering the tip deflection caused by the residual film stress in the CMOS process. The size of the microfluidic channels is 200 mm or 400 mm wide and 100 mm high. (b) Arrangement of two pairs of fully symmetric cantilevers located in two isolated microfluidic channels as a Wheatstone bridge circuit for signal read-out. The cantilevers denoted as R1 and R3 within the sensing MC channel are used to detect biochemical analytes, whereas the others denoted as R2 and R4 within the reference (Ref) channel are used for reference.

(SAM) on the gold surface of a cantilever. Then, 1 mg mL21 of BSA in PBS was injected to form a covalent link to the DTSSP through the sulfo-N-hydroxysuccinimide (sulfo-NHS) group. The immobilization process of BSA on a SAM of DTSSP was monitored in real time. Troponin I immunoassay Two monoclonal antibodies (MAbs) recognizing epitopes 41 49 (MAb 19C7) and 8690 (MAb 16A11) in troponin I (cTnI) were purchased from HyTest Ltd (Turku, Finland). cTnI is a component of the troponin complex, which consists of three different molecules: troponin C (TnC) with a molecular weight 18 kDa, cTnI with a molecular weight 23 kDa, and troponin T (cTnT) with a molecular weight 34 kDa. cTnI is released in the blood stream of a patient in the form of a binary complex with troponin C (TnC) or as a ternary cTnI-cTnT-TnC (ITC) complex.3 The ITC complex, purchased from HyTest Ltd. (Turku, Finland), was adopted in this experiment.

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Lab on a Chip

Paper force loading. Therefore, using the CAMCS design amplified the force sensitivity of the sensor at least 1250-fold. The force sensitivity can be further improved simply by increasing L/t. Compared with the physisorption of BSA, the chemisorption of BSA unto a SAM of DTSSP induced a larger and sharper signal change as shown in Fig. 4 (b). After washing with PBS twice, the signal remained at the same level (data not shown). The BSA on DTSSP is expected to reduce the conformational change of the protein compared with the direct adsorption onto a gold surface34 and yields a larger value of Dcsl . The relation between the signal change and surface properties of the cantilevers in CAMCS chips was validated by the contact angle measurement of sessile drops on Au substrates with the same modification as the cantilever surfaces (with DTSSP and BSA adsorbed on DTSSP). Based on the result of the contact angle measurement, the magnitude of signal changes in CAMCS chips correlates well with the contact angle change of the sessile drops (see Fig. S2 in the ESI3 for details). Because the contact angle is relevant to molecular-scale characterization of surfaces with dimensions of 10100 ,23 a tiny amount of BSA adsorbed on the edges of a cantilever surface can still generate a prominent contact angle change within a short period of time. The capability for force amplification and fast detection makes the CAMCS a promising solution for ultrasensitive immunoassays. CTnI detection The performance of the CAMCS chips was further demonstrated using the troponin I (cTnI) immunoassay. As in the protocol described previously, PBS solutions with various concentrations of ITC complex were injected into the CAMCS chips to detect the specific binding of cTnI. The sample injection and PBS washing generated two signal peaks resulting from the decrease in contact angle as shown in Fig. 5 (a). Lower concentrations of ITC complex (100 pg mL21) induced a smaller DVout of approximately 2 6 1023 mV compared with the 6 6 1023 mV induced by a higher dose of ITC complex (10 ng mL21), which correlates to a larger change in the solidliquid interfacial tension Dcsl from the AgAb binding. Meanwhile, the PBS washing did not cause a prominent DVout, which indicates that the mixing of two monoclonal antibodies adopted in the assays provides a higher binding affinity. Two isolated cantilever bridge sensors (BC1 and BC2) in the same CAMCS chip yielded similar signal outputs with the same ITC concentration. This finding demonstrated the reliability of the sensor design and operation. To validate the specific antigen binding in the cTnI assay, a blank experiment was performed without immobilized Ab on the cantilevers. The signal in the blank experiment changed smoothly without inducing a prominent DVout even with a higher ITC concentration as shown in Fig. 5 (a). The blank experiment also validated the success of BSA blocking on the cantilever surface because a small amount of adsorbed antigen can yield a prominent signal change as shown in the previous experiment of BSA adsorption. Based on the signal change, the detection of cTnI in the CAMCS chips generally occurs in less than 7 min, which matches the detection time of commercial instruments that are based on chemiluminescent detection with antibody-coated magnetic particles.2

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Fig. 4 (a) Characterization of the CAMCS chips. Thermal stress was induced by the self-heating of the piezoresistor to bend the cantilevers down and induce an increase in the signal output DVout of approximately 1.5 6 1022 mV. The capillary forces were induced by generating a PBS meniscus within the microfluidic channel. The initially increased signal in A in the inset is caused by the increasing liquid volume during the sample injection. (b) Signal output of the CAMCS induced by the adsorption of 1 mg mL21 of BSA in PBS on gold or DTSSP-modified gold surfaces.

the gold surface of the cantilevers, while the other is the chemisorption (immobilization) of BSA onto the DTSSPmodified gold surface. Because the adsorbed BSA made the gold surface more hydrophilic than its original state, the contact angle of the meniscus as depicted in Fig. 1 (a) will be increased by the change in the solidliquid interfacial tension Dcsl . Based on the magnitude of DVout in Fig. 4 (b), the contact angle change, Dh, could be estimated to be 6u using eqn (2)(4). Assuming that the variation of clg in PBS is negligible because of the very low concentration of BSA, the magnitude of Dcsl could then be estimated to be 22.7 mN m21 according to Dcsl ^clg cos h{ cos h. Based on the Stoney equation,13 a Dcsl of 22.7 mN m21 (and therefore, an equivalent Dss) can only generate approximately 6.4 6 1026 mV of DVout using the present cantilever design in the CAMCS chips. This signal output is approximately 1250 times smaller than the DVout from capillary

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Lab on a Chip assay was also performed to compare the data with that of the CAMCS chips (see Fig. S3 in the ESI3 for details). The standard ELISA assay and CAMCS chips demonstrated similar performance of cTnI binding below the concentration of 0.1 ng mL21. The discrepancy between the measurements using ELISA and the cantilever sensor chip of cTnI binding at higher concentrations could be explained by the sensing mechanism of the cantilever sensor depicted in Fig. 1 (a). Because the contact angle is relevant to the molecular-scale characterization of surfaces at dimensions of 10100 ,23 the cTnI that binds on the periphery of the cantilever rather than on the cantilever surface determines the contact angle change of the meniscus. Because the binding sites for cTnI on the periphery of the cantilever are much fewer than those of the entire area of the cantilever surface, the ratio of bound cTnI (on the periphery of cantilever vs. on the entire surface of cantilever) varies significantly from low to high cTnI concentration. At low cTnI concentration, the contact angle is more sensitive to the variation of cTnI binding than that at higher cTnI concentration, where most of the binding sites are already occupied. To reach the 99th percentile reference value for cTnI, detecting cTnI in the low concentration range of y0.1 ng mL21 is recommended as a cutoff to diagnose myocardial infarction by the European Society of Cardiology/American College of Cardiology (ESC/ACC).6,7 Because the change in contact angle is very sensitive to cTnI binding in minuscule amounts, as demonstrated in this work, the proposed sensing principle and cantilever sensor chip design fit very well to the current need for devices to improve the detection limit of label-free cTnI assays. In the CAMCS chips, fluidic samples remain on the top side of the cantilever surface; therefore, surface passivation on the bottom side of the cantilever, one of the important issues in the operation of conventional cantilever sensors,36 can be omitted to simplify the procedure of on-chip immunoassays. Moreover, an extra syringe pumping system is unnecessary in the CAMCS chips, which facilitates pipette-friendly operation in practical applications.

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Fig. 5 (a) Signal output of the CAMCS chips induced by the specific binding of cTnI. Higher doses of the ITC complex (10 ng mL21) induced a larger signal change in BC3 than the lower concentration of ITC complex (100 pg mL21) in BC1 and BC2 induced. A blank experiment in BC4 without immobilized Ab on the cantilever surface was performed to validate the specific binding of cTnI. (b) A dose-response of cTnI binding. The continuous line is the fifth parameter logistic (5-PL) fit for the data points. The data points for 10 ng mL21 and 0.1 ng mL21 are the overall mean of two replicate experiments conducted in two different CAMCS chips. The other data points are the mean of different cantilever sensor signals in the same CAMCS chip, error bars 1 SD. The response of non-specific binding of cTnI is plotted as a dashed line.

Conclusion
According to the magnitude of DVout, the equivalent surface stress change induced by 10 ng mL21 ITC complex is less than 10 mN m21, which is similar to the magnitude of surface stress change induced by free prostate-specific antigen (fPSA) binding with the same fPSA concentration.35,36 Instead of using a cantilever with a much larger L/t ratio to increase the detection limit, e.g., using a 600 mm long and 0.5 mm thick silicon nitride cantilever (L/t = 1200),35 the current CAMCS design (L/t = 25) can already achieve a similar detection level. The dose-response of cTnI binding, shown in Fig. 5 (b), follows a Langmuir isotherm-type behavior. The resulting curve is a fifth parameter logistic (5-PL) fit to reveal a Kd of y5 pM.37 The detection limit in the current design with the CAMCS chip (L/t = 25) is approximately 1 pg mL21. This limit is based on a noise floor of 0.5 mV pp obtained by measurement and the signal deviation caused by the non-specific binding of analytes on the blocked cantilever surface. A standard ELISA A classical method, contact angle analysis, to evaluate the molecular-scale characteristics of a solid surface was successfully integrated with a microcantilever sensor for real-time and label-free immunoassays. By converting a change in surface stress into a capillary force change through the contact angle change, the CAMCS possesses much higher force sensitivity and shorter detection times compared with the conventional approach. The proposed CAMCS chips fabricated using a CMOS process can be easily extended to a sensor array and integrated with an on-chip circuit for signal processing with the aim of becoming a POC device. Based on the CAMCS design, a further enhancement of label-free detection to the femtomolar range, which has already been achieved using fluorescence-labeling techniques, is possible by increasing the length to thickness ratio of the cantilever structure. Because of the universal mechanism of molecular recognition, the

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Lab on a Chip proposed CAMCS is expected to open a new era of real-time, label-free multiplexed biochemical assays. 15

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Acknowledgements
Published on 04 December 2012. Downloaded by Albert Ludwigs Universitaet Freiburg on 07/08/2013 15:59:25.

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This work is supported by Else Kroener-Fresenius Foundation. The authors would like to thank Chen-Fu Lin, Chun Chang, Raimar Rostek, Sebastian Wuensch, and Victor Vartanian for providing valuable input in preparing the manuscript. The authors are also grateful to the reviewers in enhancing the clarity and completeness of this paper.

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