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Label-free Detection of p53 Antibody Using A

Microcantilever Biosensor With Piezoresistive Readout

Youzheng Zhou1

, Zheyao Wang1

, Wentao Yue2

, Kai Tang2

, Wenzhou Ruan1

, Qi Zhang1

, and Litian Liu1

 1Institute of Microelectronics, Tsinghua University, Beijing 100084, CHINA

2Beijing Chest Hospital, Beijing 101149, CHINA

Email: z.wang@tsinghua.edu.cn

 Abstract  —We report the detection of p53 antibody as a

biomarker for early-stage cancer diagnosis using a

microcantilever biosensor with piezoresistive readout. The

accumulation of p53 antibody in human sera is found strongly

involved in a variety of human cancers, thus simple and fast

detection of the level of p53 antibody is of importance in clinical

application. In this work, p53 antigen is immobilized on the

surface of the microcantilever as a recognition probe to detect

p53 antibody by measuring the deflection of the microcantilever

using integrated piezoresistors, which is caused by the changes

of the surface stress as a result of the specific bioaffinity between

the antigen and the antibody. Quantitative detection of p53

antibody ranging from 20ng/ml to 20μg/ml has been achieved.

Compared with conventional ELISA or immunofluorescene

assay, the microcantilever sensor has the advantages of label-

free operation, fast detection, and low cost.

I.  I NTRODUCTION 

Diagnosis and prognosis of cancer patients with early-stage detection of convincing biomarkers is crucial for bettertreatment and higher survival rate of the patients. During the

 past decades, p53 gene, which is involved in regulating cellgrowth, replication and apoptosis, has been proved as one ofthe most important tumor suppressor by sensing DNA damage

and facilitates repair. Extensive studies have shown that themutation of p53 gene is found to associate with a wide varietyof malignant tumors including breast, lung, prostate, ovarianand melanoma [1][2]. The mutation results in the functionalteration of p53 protein from growth regulation to rapid

 proliferation of cells and accumulation of p53 protein in nucleiof tumor cells. The accumulation of p53 protein furtherinduces a higher level of p53 antibody in the serum of cancer

 patients through immune response, which has been found to be positively correlated with poor survival rate of the patients.Thus p53 antibody serves as a highly specific and effectiveclinical biomarker for early-stage cancer diagnosis and

 prognosis. Several conventional biochemical analyticalmethods such as enzyme-linked immunosorbent assay (ELISA)

or immunofluorescene assay have been applied to detect p53antibody. Each method has its inherent advantages anddisadvantages.

Microcantilevers have been emerging as sensitive andlabel-free diagnostic platform for malignant disease diagnosis

 by detecting DNA mismatch or protein biomarkers [3]-[5].The detection is normally achieved by modifying the surfaceof the microcantilever with recognition molecules. Thespecific interaction between the recognition molecules and thetarget biomarkers generates surface stress in microcantileverand in turn the deflection of the microcantilever. Thedeflection, which is normally several nanometers, can bemeasured using either optical or piezoresistive readout.

Due to the importance of p53 antibody in cancer diagnosis,we take the advantage of microcantilever biosensors tospecifically translate the molecular recognition intonanomechanical response. It is achieved by immobilizing p53antigen on the surface of the microcantilever as recognition

 probe to detect p53 antibody, the target biomarker, bymeasuring the deflection of the microcantilever, which iscaused by the changes of the surface stress as a result of thespecific bioaffinity between the antigen and the antibody. Thedeflection is measured using integrated piezoresistors, whicheliminates the bulky and expensive apparatus in common usedoptical readout. Quantitative detection of p53 antibodyranging from 20ng/ml to 20μg/ml has been achieved.Compared with conventional ELISA or immunofluoresceneassay, the present method has the advantages of label-free

operation, fast detection, and low cost.

II.  EXPERIMENTAL 

 A.   Microcantilevers

The microcantilever sensor presented here uses theintegrated piezoresistors fabricated in the single crystallinedevice layer of silicon-on-insulator (SOI) wafer, which canachieve larger piezoresistive coefficients and higher sensitivitythan polycrystalline silicon piezoresistors. Fig.1 shows theschematic illustration of the cross-section of themicrocantilever. The geometrical and fabrication parametersare optimized to increase the sensitivity of surface stresschange in protein detection [6]. The fabrication ofmicrocantilever starts with a SOI wafer with 100nm-thick p-type (100) top device layer and 400nm-thick buried oxidelayer, and the detailed process is published elsewhere [7]. The

 piezoresistors are formed by completely removing top devicelayer at other regions, and are fully encapsulated in silicondioxide dielectric layer and buried oxide layer to avoid current

This research is supported in part by the NFSC under Grant 6040009 and863 Program under Grant 2007AA03Z04.

978-1-4244-5335-1/09/$26.00 ©2009 IEEE   819 IEEE SENSORS 2009 Conference

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leakage and contamination during detection. Thin and uniformmicrocantilevers are suspended using a two-step front-sidereleasing method to achieve high efficiency and high yield [8].Owing to the total microcantilever thickness of 650nm and thelower Young’s modulus of silicon dioxide, the sensitivity ofthe present microcantilever is much larger than the formerdesign [8]. Fig.2(a) shows the SEM image of a fabricated

 piezoresistive microcantilever, and Fig.2(b) shows optical photo of the piezoresistor on the microcantilever.

Fig.1. Schematic illustration of the microcantilever with piezoresistivereadout.

(a)

Microcantilever

Piezoresistor

Lateral etching

Contact hole

Aluminum

 (b)

Fig.2. SEM image of: (a) a fabricated piezoresistive microcantilever and, (b)an on-chip piezoresistor and Wheatstone bridge configuration.

To eliminate the common mode errors such as self-heatingand vibration, two adjacent microcantilevers are operated indifferential mode during detection, i.e., one as a sensing

cantilever and the other as a reference cantilever. p53 antigenis only immobilized on the sensing cantilever, but not on thereference cantilever, thus the specific interaction is localized.The piezoresistor changes caused by the deflection are readoutusing an on-chip integrated Wheatstone bridge configuration,which consists of four piezoresistors with nominal identicalresistance value, i.e. two piezoresistors on the differential

cantilevers and two piezoresistors on substrate, as shown inFig.2(b). During the detection, the output signal of theWheatstone bridge is amplified and filtered using an off-chipinstrumental amplifier.

 B.   Functionalization

The microcantilevers are first modified using the following procedures to immobilize p53 antigen before detection.Covalent binding of recognition molecules onto solid surface

 by self-assembly monolayer (SAM) is usually used as aneffective and reliable immobilization method. 5nm chromiumand 30nm gold are successively deposited on the uppersurface of the sensing cantilever, but not on the referencecantilever. After cleaned in acetone, ethanol and deionized

water for 10min, the microcantilevers are thoroughly rinsed inan ethanol solution of 1mM 11-mercaptoundecanoic acid (11-MUA) for 12h. The mercapto-group (-SH) of 11-MUA SAMcan covalently bind on the gold surface compactly andspontaneously, and the terminal carboxy-group (-COOH) cancovalently bind with the residual amino-group (-NH3) of p53antigen by condensation reaction, thus it acts as a bridge toimmobilize the p53 antigen onto microcantilevers.

Fig. 3. Schematic view of the sensing microcantilever after functionalisation.

The terminal carboxy-group of 11-MUA SAM is activated by rinsing in the coupling reagents of 0.4M 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl)

and 0.1M N-hydroxysulfo-succinimide (NHS) for 30min toform NHS-ester [9]. Then the microcantilevers are cleaned indeionized water thoroughly to remove undesired chemicals,and the p53 antigen is then covalently immobilized byincubating in a solution of 100μg/ml p53 antigen in sodium

 phosphate buffer (PBS, 0.01M, pH=7.4) for 3h. The NHS-ester is replaced by the residual amino-group of p53 antigen tocomplete the condensation reaction. To prevent unspecific

 binding of biomarkers on the surface of the referencecantilever during detection, the microcantilevers are rinsed in15mg/ml bovine serum albumin (BSA) in PBS for 1h. After

820

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that, the microcantilevers are rinsed exhaustively in PBS toremove redundant protein molecules before detection. Thefunctionalized sensing microcantilever is schematicallyillustrated in Fig.3. The results of the SAM and theimmobilization of the recognition proteins are validated usingfluorescence imaging.

Before p53 detection, the microcantilever sensor is firstcharacterized using the bioaffinity of IgG antigen andantibody, as IgG protein pairs have been already known togenerate surface stress changes. The functionalization

 procedure follows the method aforementioned, and the IgGantibody is immobilized on the surface of the microcantileverto detect IgG antigen. After functionalization, the p53antibody detection experiment is performed in a liquidchamber. The microcantilever surface is modified using theaforementioned method, and before use the microcantileversare stabilized in PBS until the output voltage of theWheatstone bridge achieves equilibrium state. Then different

 p53 sample reagents with known concentration of p53antibodyare injected into the chamber with a flow rate of100μl/min, and the real-time voltage changes are recorded and

are translated into surface stress changes of themicrocantilevers.

III.  R ESULTS AND DISCUSSION 

Fig.4 shows a fluorescent photo of a glass carrier to verifythe SAM and the immobilization of antigen. The top leftcorner is the photo activated using 492nm laser, the top rightcorner is the photo obtained under natural light, and the

 bottom photo is the combination of the two above. It can beseen that the surface treated using EDC.HCl and NHS emitsgreen fluorescent light, whereas there is no fluorescent lightfor the area without gold. This proves that the antigen isimmobilized on the gold surface.

Fig.4 Fluorescent optical photo of the modified surface of themicrocantilever

Fig.5 shows the relative changes in the resistance versusthe concentration of IgG antigen induced by and binding

 between IgG antigen and antibody. It can be seen that thespecific bioaffinity of IgG antibody and antigen bends themicrocantilever, and the resistance changes increase with theconcentration of IgG antigen, although the relationship is not

linear. This verifies the function of bioaffinity detection of themicrocantilevers, and then they are used to detect p53antibody.

Before p53 detection, BSA with concentration of 15mg/mlis first injected as a control experiment to verify the specificityof the microcantilever. It can be seen that such a highconcentration BSA produces a small surface stress change of±2mN/m, which is negligible when compared with otherspecific bioreactions. This indicates that no specific bindingoccurs between p53 antigen and BSA.

Concentration of anti-rabbit IgG(ng/ml)

Fig.5 The relative photoresistor changes versus the antibody concentration.

Then four functionalized microcantilever sensors are usedto detect p53 antibody samples with different concentrations,namely 20ng/ml, 200ng/ml, 2μg/ml, and 20μg/ml. Themeasurement results are shown in Fig.6. It can be seen that theinjection of p53 samples impacts the microcantilever, and a

 pulse voltage occurs for a very short period (about 10s) beforethe voltage restores. For each concentration, the surface stress

increases with time and reaches a saturation stage in severalthousand seconds. The time to saturation is concentrationdependent, i.e., high concentration takes more time than lowerones. Compared with the BSA control experiment, it can beconcluded that the changes in the surface stress of themicrocantilevers are caused by the specific binding between

 p53 antigen and antibody, and the microcantilevers benddownwards.

The results show that the surface stress changes increasewith the increase of p53 antibody concentration, which meansthat quantitative detection of p53 antibody can beaccomplished in less than 20 minutes. The experiment alsogives that the limit of the detection is about 20ng/ml, which isdetermined by the fluctuation of the output signal. When p53

antibody with concentration of 2ng/ml is introduced, theoutput voltage can be distinguished from that induced byunspecific BSA.

The fluctuation and the deviation of the results mainlyoriginate from the environmental noise and variance of the

 process parameters among the microcantilevers. Therefore, inorder to improve the limit of detection, further adjustment offabrication parameters and depression of environmental noisehave to be done. 

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Fig. 5. Surface stress changes when the MCs were exposed to the samples of15mg/ml BSA and p53 antibody with concentration ranging from 20ng/ml to20 μg/ml. The MCs were stabilized before the injections at around 100s. Theturbulence signals caused by injections come back within 10s.

IV.  CONCLUSION Detection of p53 antibody is experimentally investigated

using microcantilever biosensor with piezoresistive readout.Single crystalline silicon piezoresistors are fabricated usingSOI wafers to improve the detection sensitivity. p53 antigen iscovalently immobilized on the microcantilevers as therecognition molecules via 11-MUA SAM, and detection of

 p53 antibody is studied in detail. The experiment results showthat specific and quantitative detection of p53 antibodyranging from 20ng/ml to 20μg/ml is achieved, and thedetection limit is 20ng/ml. Although the clinical detectionlimit of p53 antibody is below several ng/ml, these resultsdemonstrate that after further improvement in sensor designand fabrication, the microcantilever biosensor has the potential

in the applications of rapid and simple diagnosis of early-stagecancers.

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[4]  G. Wu, R.H. Datar, K.M. Hansen, T. Thundat, R.J. Cote and A.Majumdar, “Bioassay of prostate-specific antigen (PSA) usingmicrocantilevers,” Nat. Biotechnol., vol. 19, pp. 856–860, 2001.

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[9]   N. Patel, M.C. Davies, M. Hartshorne, R.J. Heaton, C.J. Roberts, et al.,“Immobilization of protein molecules onto homogeneous and mixedcarboxylate-terminated self-assembled monolayers,” Langmuir, vol. 13,

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